Methods and strain

ABSTRACT

The present invention relates to a method for transforming a strain of the Lactococcus genus through natural competence. The present invention further relates to strains obtained or obtainable by said method. The present invention also relates to a method for identifying a strain of the Lactococcus genus which is transformable through natural competence.

FIELD OF THE INVENTION

The present invention relates to a method for transforming a strain ofthe Lactococcus genus through natural competence. The present inventionfurther relates to strains obtained or obtainable by said method. Thepresent invention also relates to a method for identifying a strain ofthe Lactococcus genus which is transformable through natural competence.

BACKGROUND TO THE INVENTION

Lactococcus lactis is one of the most important lactic acid bacteriaused in the dairy industry, in particular as a main dairy starterspecies in various cheese preparations (e.g. gouda, cheddar, brie,parmesan, roquefort) and fermented milk products (e.g. buttermilk, sourcream). Other applications of L. lactis bacteria include as a host forheterologous protein production or as a delivery platform fortherapeutic molecules. While the growth and fermentation properties ofL. lactis have been gradually improved by selection and classicalmethods, there is great potential for further improvement throughnatural processes or by genetic engineering. Of particular interest aremethods to naturally transform L. lactis without the use of geneticengineering, thereby generating new non-GMO strains with usefulindustrial properties.

Lactococcus raffinolactis is present in a wide range of environments,such as foods (meat, fish, milk, vegetable), animals, and plantmaterials. In the dairy environment, this species has been found in rawmilks (cow, ewe, goat, and camel), natural dairy starter cultures, and agreat variety of cheeses. The prevalence of this bacterium in foods evenif with a “nondominant” status compared to other lactococci could makeit a candidate for future development of starter cultures.

DNA acquisition by natural transformation is widespread amongprokaryotes and has been identified in over 80 species. Variousfunctions are attributed to competence for natural transformation:genome plasticity, DNA repair, and/or nutrition. In Gram-positivebacteria, competence for natural transformation has beenwell-characterized in Bacillus subtilis and in various species of thegenus Streptococcus (e.g. S. pneumoniae, S. mutans, and S.thermophilus).

In streptococci, competence for DNA transformation is induced inresponse to secreted signalling peptides referred to as competencepheromones/alarmones. The production of this class of cell-to-cellcommunication molecules is initiated in response to specificenvironmental stresses or conditions and allows the coordination ofphysiological functions (e.g. competence, predation, biofilm formation).Above a threshold concentration, competence pheromones activate themaster regulator ComX (alternative sigma factor σ^(X)), which ultimatelyleads to a transcriptional reprogramming of cells (globally known aslate competence phase) including the induction of genes strictlyrequired for DNA transformation. ComX binds to a specific DNA sequencenamed Com-box or Cin-box, which is located at least in the vicinity ofpromoters of late competence (corn) genes/operons responsible for DNAuptake (e.g.; comG, comF and comE operons), DNA protection (e.g. ssb)and DNA recombination (e.g. recA, dprA, coiA), and positively controlstheir expression.

The early steps leading to competence activation (early competencephase) differs among bacteria. In streptococci, two major peptide-basedsignaling pathways—i.e. ComCDE and ComRS—have been identified so far. Inmitis and anginosus groups of streptococci (S. pneumoniae as paradigm),the competence signaling peptide (CSP, or mature ComC) triggers aphosphorylation cascade mediated by the two-component system ComD-ComE,leading to the transcriptional activation of comX. In salivarius,mutans, pyogenes, bovis and suis groups of streptococci, anotherregulation mechanism is operational (S. thermophilus as paradigm). Thissystem involves the ComX-induction peptide (XIP, or mature ComS) whichis internalized by the oligopeptide transporter Opp, binds to andactivates the regulator ComR, and in turn induces comX transcription.

Orthologues of comX and of all late corn genes essential for naturaltransformation have been identified in the genome of L. lactis, althoughsome are present as putative pseudogenes in different strains (Wydau etal., 2006).

Specific growth conditions have been reported to activate corn genes inLactococcus lactis. For example, the promoter of comX was shown to beinduced during cheese-making conditions in strain MG5267 (an MG1363derivative) which belongs to the subspecies cremoris (Bachmann et al.2010).

In the L. lactis subspecies (subsp.) lactis, carbon starvation was shownto activate six late corn genes in strain IL1403 of dairy origin (i.e.comX, comEA, comGA, comGB, radA, and nucA) and most of the lateessential corn genes in strain KF147 of plant origin (i.e. comX, comC,coiA, and operons comG, comE, comF) (Ercan et al., 2015). However, whenthe authors attempted to validate functional natural transformation inKF147, they were unsuccessful.

Wydau et al. reported that all the well-established late genes/operonsdisplay an upstream and conserved Com-box, suggesting that they aresimilarly controlled by ComX as reported in streptococci. However, theauthors did not comment on whether comX over-expression in IL1403induced natural competence. Indeed, the authors neither report anyexperiment evaluating natural competence in this strain nor suggest anyexperimental conditions appropriate for inducing natural competence.Thus, as noted in the recent literature (see Ercan et al., 2015) [i.e.,9 years after Wydau et al.], there is no experimental evidence forsuccessful transformation of any species of the genus Lactococcus bynatural competence, and even less of IL1403.

Accordingly, there remains a need for a method for naturallytransforming Lactococcus strains using natural competence. In addition,since some strains of the Lactococcus genus may not encode a full set offunctional late corn genes, there is a need for a method for identifyingLactococcus strains which can be transformed by natural competence.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method fortransforming a strain of the Lactococcus genus with an exogenous DNApolynucleotide comprising the steps of:

-   -   (a) providing a strain of the Lactococcus genus, wherein said        strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.

In one embodiment, the step of modulating the production of a ComXprotein is performed by expressing a comX gene in said strain orincreasing the expression of a comX gene in said strain.

In a further embodiment, the comX gene is an exogenous comX gene. Saidexogenous comX gene may be transferred into said strain by conjugation,transduction, or transformation. Said exogenous comX gene may beoperably linked to transcription regulator(s).

In an alternative embodiment, said comX gene is the endogenous comX geneof said strain.

In one embodiment, when said comX gene is the endogenous comX gene ofsaid strain, the method comprises:

-   -   (a) providing a strain of the Lactococcus genus, wherein said        strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein, by expressing        the endogenous comX gene or increasing the expression of the        endegenous comX of said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome,    -   wherein step (c) is carried out after step (b) or wherein        step (b) and step (c) are carried out simultaneously.

In some embodiments, said ComX protein has the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, or has at least 90% identity or at least 90% similarity to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20 or SEQ ID NO:22. In some embodiments, said ComXprotein has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, or has at least 90% identity or at least 90% similarity to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

In some embodiments, said comX gene has the nucleotide sequence of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, or has at least 90% identity to the nucleotide sequence of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21.

In some embodiments, said comX gene has the nucleotide sequence of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, or has at least 90% identity to thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

In some embodiments, the medium of step (c) is a chemically definedmedium. In a preferred embodiment the chemically defined medium (CDM)comprises 0.5 g/L NH₄Cl, 9.0 g/L KH₂PO₄, 7.5 g/L K₂HPO₄, 0.2 g/L MgCl₂,5 mg/L FeCl₂, 50 mg/L CaCl₂), 5 mg/L ZnSO₄, 2.5 mg/L CoCl₂, 0.05 g/Ltyrosine, 0.1 g/L asparagine, 0.1 g/L cysteine, 0.1 g/L glutamine, 0.1g/L isoleucine, 0.1 g/L leucine, 0.1 g/L methionine, 0.1 g/L tryptophan,0.1 g/L valine, 0.1 g/L histidine, 0.2 g/L arginine, 0.2 g/L glycine,0.2 g/L lysine, 0.2 g/L phenylalanine, 0.2 g/L threonine, 0.3 g/Lalanine, 0.3 g/L proline, 0.3 g/L serine, 10 mg/L paraaminobenzoic acid,10 mg/L biotin, 1 mg/L folic acid, 1 mg/L nicotinic acid, 1 mg/Lpanthotenic acid, 1 mg/L riboflavin, 1 mg/L thiamine, 2 mg/L pyridoxine,1 mg/L cyanocobalamin, 5 mg/L orotic acid, 5 mg/L 2-deoxythymidine, 5mg/L inosine, 2.5 mg/L dl-6,8-thioctic acid, 5 mg/L pyridoxamine, 10mg/L adenine, 10 mg/L guanine, 10 mg/L uracil, 10 mg/L xanthine, and 5g/L glucose.

In some embodiments, prior to step (c) said strain is incubated in apre-culture medium, preferably wherein the pre-culture medium is acomplex medium, more preferably wherein the pre-culture medium is M17Gor THBG.

In some embodiments of the present invention, said strain is incubatedwith the exogenous DNA polynucleotide for around 4 to 8 hours at around30° C. and said medium of step (c) is supplemented with anosmo-stablizer, preferably wherein the osmo-stablizer is glycerol ormannitol, more preferably wherein the osmo-stabilizer is 5% [v/v]glycerol or 5% [w/v] mannitol.

In some embodiments, said exogenous DNA polynucleotide is from a strainof the Lactococcus lactis species.

In some embodiments, said exogenous DNA polynucleotide is from a strainof the Lactococcus raffinolactis species.

In some embodiments, said strain of step (a) is a Lactoccocus lactissubsp. cremoris strain.

In another aspect, the present invention provides a strain of theLactococcus genus obtained or obtainable by the method of the firstaspect of the present invention.

In one embodiment, said strain of the Lactococcus genus is a strain ofthe Lactococcus lactis or Lactococcus raffinolactis species.

In a further aspect, the present invention provides a method foridentifying a strain of the Lactococcus genus which is transformablethrough natural competence comprising the steps of:

-   -   (a) providing a strain of the Lactococcus genus species;    -   (b) transforming said strain with a plasmid expressing a comX        gene having at least 90% identity, preferably having 100%        identity, to the endogenous comX gene of said strain;    -   (c) contacting said strain obtained in step (b) with an        exogenous DNA polynucleotide encoding a marker gene in a medium        and incubating the resulting mixture for integration of the        exogenous DNA polynucleotide into the genome of said strain; and    -   (d) determining the rate of recombination events;

wherein a rate of at least 1×10⁻⁶ transformants per μg of DNA isindicative of a strain which is transformable through naturalcompetence.

In a particular embodiment of method for transforming a strain of theLactococcus genus of the present invention, said strain of step (a) isidentified using the method for identifying a strain of the Lactococcusgenus which is transformable through natural competence according to thepresent invention. In some embodiments of the present invention, saidstrain of step (a) is identified using Assay A.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Table showing the status of genes involved in natural competencefor L. lactis strains MG1363, SK11, KW2, IL1403, SL12651 and SL12653.

Late corn genes in the complete genomes of strains MG1363, SK11, and KW2of Lactococcus lactis subsp. cremoris and of strain IL1403, SL12651 andSL12653 of Lactococcus lactis subsp. lactis. Origin is indicated abovestrain names. Gene-associated function in DNA transformation isindicated on the left. Reg. denotes regulation. The complete andincomplete status of late genes is based on blastp and tblastn homologysearches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using orthologues ofS. pneumoniae TIGR4 and S. thermophilus LMD-9 and default parameters.+denotes the presence of a complete gene; * denotes the presence of anincomplete gene due to nucleotide(s) exchange, insertion or deletionresulting in a premature stop codon; and Tn denotes a disrupted gene bythe insertion of at least one transposon.

FIG. 2: Graphs displaying the results of luciferase assays whichdemonstrate the activation of a reporter construct comprising the latepromoter P_(comGA) driven by constitutive comX overexpression

(A) Maximum specific luciferase (Lux) activity (RLU OD₆₀₀ ⁻¹) emitted byeight independent clones (cl01 to cl08) of the KW2-derived reporterstrain (BLD101, P_(comGA[MG]-lux)AB) carrying plasmid pGhP32comX_(MG)compared to the control strain (Ctl) carrying the empty vector pG⁺host9.(B) Kinetics of specific Lux activity (solid line) during growth(RLU/OD₆₀₀; dotted line) for the control strain (Ctl; black lines) andthree selected clones (BLD101 [pGhP32comX_(MG)], cl02, cl04 and cl05;gray lines). (C) Kinetics of specific luciferase activity (closedsymbols) during growth (RLU/OD₆₀₀; open symbols) of theMG1363+pGhP32comX_(MG)-P_(comGA[MG])-luc, grown in M17G at 30° C. (D)Kinetics of specific luciferase activity (closed symbols) during growth(RLU/OD₆₀₀; open symbols) of IL1403+pGhP32comX_(IO)-P_(comGA[IO])-lucstrains, grown in M17G at 30° C.

FIG. 3: Graphs displaying the results of luciferase assays whichdemonstrate the impact of growth medium on P_(comGA) activation

Maximum specific Lux activity of BLD101 [pGhP32comX_(MG)] cl02 grown indifferent final culture media (CDM, THBG, and M17G) according topreculture conditions (CDM, THBG, and M17G). Overnight precultures were10-fold diluted in the pre-culture medium and grown for 2 hours. Then,cells were washed twice in distilled water and the OD₆₀₀ was adjusted to0.05 in the final growth medium before measuring growth and luciferaseactivity. One representative experiment of two independent replicates.

FIG. 4: Results of a transformation assay implemented on a L. lactissubsp. cremoris KW2 constitutively expressing comX contacted with a DNAconsisting of a mutated allele of the rpsL gene as exogenous DNApolynucleotide

(A) Alignment of the rpsL gene sequences of strain MG1363, a spontaneousstreptomycin-resistant clone of strain MG1363, strain KW2 and aKW2-derived transformant obtained using the method of the invention(partial sequence). The arobase, pound and dollar signs below thealignment indicate the positions of nucleotide differences existingbetween the rpsL sequences. The dollar sign at position 167 indicatesthe point mutation (A→T; strA1 allele) responsible for thestreptomycin-resistance phenotype; the pound sign at position 156highlights a nucleotide that is naturally different between MG1363 andKW2 (T in KW2, A in MG1363); the arobase sign at position 39 indicates asilent nucleotide substitution (T→G) which is found in thestreptomycin-resistant clone derived from MG1363. (B) DNA transformationwith the strA1 allele was assessed for L. lactis strains constitutivelyexpressing ComX. Transformation rate (white bars) and maximum specificluciferase (Lux) activity (black diamonds, RLU OD₆₀₀ ⁻¹, as reported inFIG. 2) of eight clones (cl01 to cl08) of the reporter strain (BLD101,P_(comGA[MG])-luxAB) carrying plasmid pGhP32comX_(MG) compared to thenegative control strain (Ctl−) carrying the empty vector (BLD101[pG⁺host9]).

FIG. 5: Graphs displaying the results of transformation rate of the KW2derivative BLD101 [pGhP32comX_(MG)] obtained with overlap PCR products(comEC, mecA, ciaRH, covRS and clpC) and strA1 (rpsL*)-donor DNA.

The threshold represents the theoretical transformation rate to obtainonly one transformant.

FIG. 6: Graphs depicting the results of transformation assays for a L.lactis subsp. cremoris deleted in its comEC gene and constitutivelyexpressing comX.

DNA transformation with the strA1 allele was assessed for L. lactisstrains constitutively expressing comX. Transformation rate (white bars)and maximum specific luciferase (Lux) activity (RLU OD₆₀₀ ⁻¹) of fourclones (cl01 to cl04) of the ComEC-deficient reporter strain (BLD102,P_(comGA[MG])-luxAB) carrying plasmid pGhP32comX_(MG) compared to thepositive (Ctl⁺, BLD101 [pGhP32comX_(MG)] cl02) and negative (Ctl⁻,BLD101 [pG⁺host9]) control strains. Transformability was assessedaccording to the standard protocol described in Materials and Methodsusing strA1-carrying PCR products as donor DNA. ND denotes atransformation rate below the detection level of spontaneous Str^(r)mutants (<10⁻⁷). One representative experiment of two independentreplicates.

FIG. 7: Graphs displaying natural competence in Lactococcus lactissubsp. lactis SL12651 and 12653 strains.

(A) Transformation rate of L. lactis subsp. lactis SL12651 and 12653strains in M17G medium, with rpsL* donor DNA (+DNA) or without donor DNA(−DNA); (B) DNA transformation with increasing initial concentration ofdonor DNA assessed in SL12653 strain; (C) Comparison of transformationrates between wild-type (WT) SL12653 strain and a SL12653 strain deletedfor the comX gene (ComX−); transformation rate of three clones of theComX-deficient strain compared to the WT strain, in presence (+DNA) orin absence (−DNA) of donor DNA.

DETAILED DESCRIPTION

The present invention is based on the observation that overexpression ofComX in a strain of the Lactococcus genus allowed to transform thisstrain by natural competence. Using this approach a L. lactis strain wasgenerated by natural transformation with an exogenous DNApolynucleotide. Importantly, these results are the first demonstrationof transformation of a L. lactis strain by natural competence. Further,existence of natural competence in the Lactococcus genus has beenconfirmed in two strains of the Lactococcus raffinolactis species andtwo Lactococcus lactis species.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, biochemistry,microbiology, bacteriology, and related fields, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature.

Thus, the present invention provides a method for transforming a strainof the Lactococcus genus with an exogenous DNA polynucleotide comprisingthe steps of:

-   -   (a) providing a strain of the Lactococcus genus, wherein said        strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.

As detailed below, step (b) and step c) can be carried out sequentially[i.e., step (b) and then step (c)] or in another embodiment step (b) andstep (c) can be carried out simultaneously.

Lactococcus Genus

The present invention relates to a method for transforming a strain ofthe Lactococcus genus, a Gram-positive bacterium. Lactococcus strainsare known as lactic acid bacteria (LAB) for their ability to convertcarbohydrate to lactic acid. A strain of the Lactococcus genus andLactococcus strain are used herein interchangeably.

The Lactococcus genus comprises, but is not limited to the followingspecies: Lactococcus chungangensis, Lactococcus fujiensis, Lactococcusgarvieae, Lactococcus lactis, Lactococcus piscium, Lactococcus plantarumand Lactococcus raffinolactis. Any strain of one of these species may beused in the current invention, provided that this strain istransformable through natural competence as defined herein.

In a particular embodiment, said strain of the Lactococcus genus of stepa) is a strain of the Lactococcus lactis species or a strain of theLactococcus raffinolactis species.

Lactococcus lactis

In a particular embodiment, said strain of the Lactococcus genus of stepa) is a strain of the Lactococcus lactis species. The speciesLactococcus lactis comprises several subspecies. Thus, when the strainof the Lactococcus genus of step a) is a strain of the Lactococcuslactis species, said strain is selected in the group consisting ofLactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae,Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. tructae.As used herein a strain of the Lactococcus lactis species is understoodto be a genetic variant or subtype of any L. lactis species orsubspecies. The different Lactococcus lactis subspecies disclosed here,and in particular the lactis and the cremoris subspecies, are definedherein based on DNA sequences coding for 16S ribosomal RNA [Ward et al.,1998].

In a particular aspect, the present invention provides a method fortransforming a strain of the Lactococcus lactis species with anexogenous DNA polynucleotide comprising the steps of:

-   -   (a) providing a strain of the Lactococcus lactis species,        wherein said strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.

In a preferred embodiment, the strain of step (a) is a Lactococcuslactis subsp. cremoris strain or a Lactococcus lactis subsp. lactisstrain. Both subspecies have been identified and characterised with fullgenome sequences see, e.g., Wegmann et al. (2007) J. Bacteriol.189:3256-3270 and Bolotin et al. (2001) Genome Res. 11:731-753. Withregards to the dairy industry, L. lactis subsp. lactis (previously knownas Streptococcus lactis) is preferred for making soft cheese while L.lactis subsp. cremoris (previously known as Streptococcus cremoris) ispreferred for hard cheese production.

In a preferred embodiment, the strain of step (a) is Lactococcus lactissubsp. cremoris strain.

In another preferred embodiment, the strain of step (a) is Lactococcuslactis subsp. lactis strain.

Lactococcus raffinolactis

In a particular embodiment, said strain of the Lactococcus genus of stepa) is a strain of the Lactococcus raffinolactis species.

In a particular aspect, the present invention provides a method fortransforming a strain of the Lactococcus raffinolactis species with anexogenous DNA polynucleotide comprising the steps of:

-   -   (a) providing a strain of the Lactococcus raffinolactis species,        wherein said strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.        Lactococcus plantarum

In a particular embodiment, said strain of the Lactococcus genus of stepa) is a strain of the Lactococcus plantarum species.

In a particular aspect, the present invention provides a method fortransforming a strain of the Lactococcus plantarum species with anexogenous DNA polynucleotide comprising the steps of:

-   -   (a) providing a strain of the Lactococcus plantarum species,        wherein said strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.        Lactococcus piscium

In a particular embodiment, said strain of the Lactococcus genus of stepa) is a strain of the Lactococcus piscium species.

In a particular aspect, the present invention provides a method fortransforming a strain of the Lactococcus piscium species with anexogenous DNA polynucleotide comprising the steps of:

-   -   (a) providing a strain of the Lactococcus piscium species,        wherein said strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.        Lactococcus garvieae

In a particular embodiment, said strain of the Lactococcus genus of stepa) is a strain of the Lactococcus garvieae species.

In a particular aspect, the present invention provides a method fortransforming a strain of the Lactococcus garvieae species with anexogenous DNA polynucleotide comprising the steps of:

-   -   (a) providing a strain of the Lactococcus garvieae species,        wherein said strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.        Lactococcus fujiensis

In a particular embodiment, said strain of the Lactococcus genus of stepa) is a strain of the Lactococcus fujiensis species.

In a particular aspect, the present invention provides a method fortransforming a strain of the Lactococcus fujiensis species with anexogenous DNA polynucleotide comprising the steps of:

-   -   (a) providing a strain of the Lactococcus fujiensis species,        wherein said strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.        Lactococcus chungangensis

In a particular embodiment, said strain of the Lactococcus genus of stepa) is a strain of the Lactococcus chungangensis species.

In a particular aspect, the present invention provides a method fortransforming a strain of the Lactococcus chungangensis species with anexogenous DNA polynucleotide comprising the steps of:

-   -   (a) providing a strain of the Lactococcus chungangensis species,        wherein said strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain of step (b) with an exogenous DNA        polynucleotide in a medium and incubating the resulting mixture        for integration of the exogenous DNA polynucleotide into the        genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.

DNA Acquisition

Bacteria may naturally acquire exogenous DNA via one of three possiblemechanisms: transformation, conjugation, or transduction.

As used herein the term “transformation” refers to the uptake ofexogenous genetic material (e.g. a DNA polynucleotide) from the externalmedium. Since transformation requires that genetic material cross thebacterial cell wall and membrane and the uptake of exogenous geneticmaterial is energetically costly, the process is tightly regulated.Accordingly, bacterial cells may only be transformed under certainconditions. Bacterial cells which are in a transformable state are saidto be competent.

Competence may be artificially induced in the laboratory, e.g. byelectroporation or exposure to divalent cations (e.g. CaCl₂)) and heatshock. Alternatively, some species of bacteria express a proteinaceousmachinery that provides natural competence; this system of naturalcompetence has been widely studied in streptococci.

As used herein the term “conjugation” refers to the transfer of geneticmaterial between bacterial cells.

As used herein the term “transduction” refers to the transfer of geneticmaterial from a virus (e.g. a bacteriophage) or a viral vector intobacterial cell.

ComX Protein

The method of the present invention comprises the step of modulating theproduction of a ComX protein in said strain.

ComX protein is an alternative sigma factor, also known as σ^(x), whichacts as master regulator for the late corn genes and is responsible fortranscriptional reprogramming of cells including the induction of genesstrictly required for DNA transformation (Lee et al., 1989; Petersen etal. 2004).

ComX may bind to a specific target sequence (or box) termed the Com-box(or Cin-box). Com-boxes are located in the vicinity of the promoters oflate competence (corn) genes/operons responsible for DNA uptake (e.g.,comG, comF, and comE operons), DNA protection (e.g. ssb) and DNArecombination (e.g. recA, dprA, coiA), and positively controls theirexpression (Campbell et al., 1998; Luo and Morrison, 2003).

The production of the ComX protein in a strain of interest may beincreased relatively to an appropriate control strain, i.e., theLactococcus strain in which the production of the ComX protein has notbeen modulated. ComX protein may be produced (expressed) followingmodulation as compared to an appropriate control strain, i.e., theLactococcus strain in which the ComX protein is not produced.

In some embodiments, the production of the ComX protein is constitutiveor inducible.

The production of ComX protein may be monitored using any method knownin the art. For example, by western blotting using an antibody specificfor the ComX protein. Alternatively, comX gene mRNA transcript levelsmay be measured by qPCR.

Alternatively, the ComX protein may be monitored using a reporterconstruct polynucleotide, e.g. as described in the Example 1 andMaterials and Methods. The reporter construct polynucleotide maycomprise genes encoding one or more reporter proteins, preferably thegenes encoding the reporter proteins are operably linked to a promotercomprising a Com-box sequence. The reporter proteins may be LuxAB orLuc. Accordingly, ComX expression (and activity) may be detected andmeasured using a luciferase assay (Fontaine et al., 2010).

In some embodiments of the method of the present invention, the step ofmodulating the production of a ComX protein is performed by expressing acomX gene in said strain or increasing the expression of a comX gene insaid strain. In a particular embodiment, the step of modulating theproduction of a ComX protein is performed by expressing a comX gene insaid strain in some growth conditions, whereas said strain does notexpress the ComX protein outside of these growth conditions. In aparticular embodiment, the step of modulating the production of a ComXprotein is performed by increasing the expression of a comX gene in saidstrain in some growth conditions.

The comX gene may be an exogenous comX gene. As used herein an“exogenous comX gene” is understood to be a comX gene which is broughtinto the cytoplasm of the Lactococcus strain of step a), in order to beexpressed. The exogenous comX gene may have the same sequence as thecomX gene found in the genome of the Lactococcus strain of step a) ormay have a different sequence from the comX gene found in the genome ofthe Lactococcus strain of step a). When different, the comX gene may bederived from a strain of a different species, a different subspecies ora different strain of Lactococcus.

The exogenous comX gene may be integrated within the genome of saidLactococcus strain.

Alternatively, the exogenous comX gene may be located within a vector.The vector may be selected from a plasmid, a viral vector (e.g. aphage), a cosmid, or a bacterial artificial chromosome.

Said plasmid may be transferred into said Lactococcus strain byconjugation, transformation or transduction. Said plasmid may beauto-replicative in the transformed Lactococcus strain or not.

The exogenous comX gene may be operably linked to transcriptionregulator(s). The exogenous comX gene may be located in a linear orcircular polynucleotide.

Alternatively, in some embodiments of the method of the presentinvention, the comX gene is the endogenous comX gene of said Lactococcusstrain. As used herein “the endogenous comX gene of said strain” isunderstood to be a comX gene that is naturally present in the genome ofsaid strain.

In some embodiments, said comX gene is a Lactococcus comX gene. In anembodiment, said comX gene is a Lactococcus lactis comX gene. In aparticular embodiment, said comX gene is a Lactococcus lactis subsp.lactis comX gene. In a particular embodiment, said comX gene is aLactococcus lactis subsp. cremoris comX gene.

The comX gene may comprise or consist of a nucleotide sequence selectedfrom the group consisting of:

-   -   SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,        SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID        NO:19; SEQ ID NO:21;    -   a nucleotide sequence having at least 90%, at least 91%, at        least 92%, at least 93%, at least 94%, at least 95%, at least        96%, at least 97%, at least 98%, or at least 99% identity to the        nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,        SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID        NO:15, SEQ ID NO:17, SEQ ID NO:19; SEQ ID NO:21;    -   a variant of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,        SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID        NO:17, SEQ ID NO:19, SEQ ID NO:21 encoding respectively a ComX        protein of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,        SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID        NO:18, SEQ ID NO:20 or SEQ ID NO:22; and    -   a variant of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,        SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID        NO:17, SEQ ID NO:19, SEQ ID NO:21 encoding respectively a        functional ComX protein having at least 90% identity or at least        90% similarity to a ComX protein of SEQ ID NO:2, SEQ ID NO:4,        SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID        NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.

[SEQ ID NO: 1]ATAACATATTACTTGGAAGAAGAGGATTTTGAAAATCTTTTTTCAGAAATGAAACCTATAGTTATGAAATTAATGAAACAAATTCGCATTAGAACATGGAAAATAGAGGATTATCTTCAAGAGGGGATGATTATTTTACATCTTCTATTAGAAGAGCAGAACGATGGTCAAAAGCTGCATACAAAATTTAAGGTAAAGTATCATCAAAGATTAATAGATGAATTAAGACGAAGTTATGCAAAGAAACGAAGCCATGACCATTTTATAGGTTTAGATGTTTATGAATGCTCAGACTGGATAAATTCAGGTGATACTAGTCCAGATAATGAAGTGGTCTTCAATCATTTGCTGGCAGAAGTATATGAAGGTTTGAGCGCACATTATCAAGACTTACTACTTCGACAAATGCGAGGAGAAGAACTAACTCGCATGCAACGGTATCGCCTTCGTGAAAAAATAAAGGCCATCTTATTTTCAGAAGACGAAGAGTGA [SEQ ID NO: 2]MTYYLEEEDFENLFSEMKPIVMKLMKQIRIRTWKIEDYLQEGMIILHLLLEEQNDGQKLHTKFKVKYHQRLIDELRRSYAKKRSHDHFIGLDVYECSDWINSGDTSPDNEVVFNHLLAEVYEGLSAHYQDLLLRQMRGEELTRMQRYRLREKIKAILFSEDEE [SEQ ID NO: 3]ATGACATATTACCTGGAAGAAAATGAATTCGAAGGTTTATTTTCTGGAATGAAACCAATCATCAGAAAATTGATGAAACAAATTCGAATCAAAGCATGGGACATAGAGGATTATTATCAAGAAGGAATGATTATTTTGCATCACCTTTTAGAAGAAAATCACCCATCCACTAATATTTATACAAAGTTCAAAGTAAAATATCATCAACATTTGATTGATGAACTACGCCATAGCTACGCCAAAAAACGGCTTCATGACCATTTTGTAGGTCTGGACATTTATGAATGTTCGGACTGGATAGATGCAGGAGGAAGTACCCCTGAAAGCGAGCTTGTGTTCAATCATCTTTTAGCAGAAGTTTATGAAGGATTGAGCGCCCACTATCAGGAATTACTCGTGCGTCAAATGAGAGGAGAAGAACTCACGCGAATGGAACGCTATCGGCTAAGAGAAAAAATCAAAAATATACTATTTTCTCGAGATGATGATTAA [SEQ ID NO: 4]MTYYLEENEFEGLFSGMKPIIRKLMKQIRIKAWDIEDYYQEGMIILHHLLEENHPSTNIYTKFKVKYHQHLIDELRHSYAKKRLHDHFVGLDIYECSDWIDAGGSTPESELVFNHLLAEVYEGLSAHYQELLVRQMRGEELTRMERYRLREKIKNILFSRDDD [SEQ ID NO: 5]ATGGATGACATTCAAGAAAAATACGGTTTAGAATTCAACGAATTATTCTCTGAGATGCGGCCGATAATTTATAAATTGATGAAGCAATTGCACATCAACACATGGGATTACGATGATTACTTCCAAGAGGGAATGATTACACTACATGAATTGCTGCAGAAAATTACAAATTTAGATCATGTACATACGAAATTTAAAGTGGCTTACCATCAGCACTTAATTGACGAAATTCGCCATATTAAAGCACGAAAAAGAGGTTTTGATCAGCTCCATCCGATCAATGTTTATGACTGCGCAGATTGGATTGGCTCAAACCTTGCTACACCTGAAAGCGAGATAGTTTTCAACCATCTACTAGAAGAAGTTTATGATAAACTTTCAACACACTATAAAGAACTGTTGGTAAAGCAAATGCATGGGGAACATCTTACGAGAATGCAGAAGTATCGTTTAAAGGAAAAAATTAAAGCGATTTTATTTGATGAAGACTAA [SEQ ID NO: 6]MDDIQEKYGLEFNELFSEMRPITYKLMKQLHINTWDYDDYFQEGMITLHELLQKITNLDHVHTKFKVAYHQHLIDEIRHIKARKRGFDQLHPINVYDCADWIGSNLATPESEIVFNHLLEEVYDKLSTHYKELLVKQMHGEHLTRMQKYRLKEKIKAILFDED [SEQ ID NO: 7]ATGGATAAAATTGAAACCATACTTAAAAGTATTGAACCGATTATTATGAACTGTCGGAAAAAAACTAAAATTCCTTCCTGGGAATTAGACGACTATATGCAGGAAGGGATGATTATTGCTTTAGAGATGTACCATCAACTCTTATTAGATCCACCAGATGATGACTTTAACTTCTATGTCTATTTCAAAGTCAGGTATTCTTGTTTCTTAATTGATCACTATCGCAAAGCTATGGCAGTCAAGAGAAAATTCGACCAGCTTGACTATTGTGAACTTTCTGAGTCTGTTAATCTTTTTGATCACAAACAAAATGTGTCTGAAAACGTCATGTATAACTTGTTGTGTCAAGAAATACACTTGGTTTTATCCCCGGAGGAGCTCAAGCTTTTTGAGGCACTTATTTGA[SEQ ID NO: 8]MDKIETILKSIEPIIMNCRKKTKIPSWELDDYMQEGMIIALEMYHQLLLDPPDDDFNFYVYFKVRYSCFLIDHYRKAMAVKRKFDQLDYCELSESVNLFDHKQNVSENVMYNLLCQEIHLVLSPEELKLFEALI[SEQ ID NO: 9]ATGGATAGCATAGAAATGATGCTTCAAAATATTGAGCCAATTATTATGAATTGTAGTAAAACAACTAGGATTCCATCTTGGGAGCTAGATGATTACATGCAGGAGGGGATGATTATTGCACTGGAAATGTATCAAAATAGACATAACATCAATAACGGTAACGCGTTTAATTTCTATGTCTATTTTAAAGTCAGGTATTCCTGTTACCTGATAGATAGTTTTAGAAAGGCTAACGCATATAAAAGAAAATTTGATCAACCATTATATTGTGAAATATCTGAAGCCTTCAACCTTTATGATCACCACCAAAATGTTGCAGACAATGTCTGTTATCAGCTATTGCAAGTTGAAATTCTTGAGATATTAACACCAGATGAAGCTGATTTATTTATGACCTTGAAAAATGGTGGGAAAGTAGAGAGAAATAAAAAGTATAGATTAAAGAAAAAAATTATTGATTATCTTAAAGACATGTTATGA [SEQ ID NO: 10]MDSIEMMLQNIEPIIMNCSKTTRIPSWELDDYMQEGMIIALEMYQNRHNINNGNAFNFYVYFKVRYSCYLIDSFRKANAYKRKFDQPLYCEISEAFNLYDHHQNVADNVCYQLLQVEILEILTPDEADLFMTLKNGGKVERNKKYRLKKKIIDYLKDML [SEQ ID NO: 11]ATGGAGACTTTAGAAGCCATGCTCAAAAACATTGAACCTATTATTATGAATTGTCAAAAGATGGCAAAAATACCTTCCTGGGATATTGACGATTATATGCAGGAGGGGAGGATCATTGCATTAGACTTGTATAATCAGCTAGCAGAAAGAATGGAGACGGATGAGGTGAACTTTTACGTCTACTTCAAAGTCAGATATACCTGTTTCTTGATTGATACTTACCGTAAGACAAATGCCTTTAAAAGAAAATTTGACCAACCGATTTACTTAGATGTATCCGAAGCATTTAATCTGTATGATCATAAGCAGAATGTCGCTGATAATGTCATGTATACTTTATTGCATCAGGAGATTCTAGACATCTTAACGCCTGTAGAAATTCAAACGCTAAACGCACTAAAAAGGGGAGAAAAGGTCGACCGCAATAAAAAATTTAGGATTAAAAAGAAGATTATCAACTATATTAATCAGATTTTCTAG [SEQ ID NO: 12]METLEAMLKNIEPIIMNCQKMAKIPSWDIDDYMQEGRIIALDLYNQLAERMETDEVNFYVYFKVRYTCFLIDTYRKTNAFKRKFDQPIYLDVSEAFNLYDHKQNVADNVMYTLLHQEILDILTPVEIQTLNALKRGEKVDRNKKFRIKKKIINYINQIF [SEQ ID NO: 13]ATGGAGCATAATTTAGATATGGAGCAGCTGGAAGAAATTTTTCATTCTGTCCAACATATTGTGTGGAAGAACAGTCGTTTGATTCCGATAAATTTTTGGACGTTTGATGACTATCAGCAGGAAGGGCGCTTGGTATTATACGATTTGCTGGGAGATGGTGTGACGCAAAGGAACTTATTTTGCCATTTTAAGGTACGCTATAAGCAGAGACTTATTGATATTAAAAGAAGGGAGCGGGCTTTTAAAAGGGGTTTTGATTGCGGGACTGGCTTAGATATATACGAATATTCTGATGCTCTAAAGGGGAAAGCAGCCAGTCCAGAACATATCCTGATTTCTGGAAGTTTACTTGAAGAAGTTTTTGAAAACTTAAATTTACGCTACCGACGGCTCCTCAAAAGTTACCTCGCCGGCGATGAATTGCACCGTATGGAAAAGTATCGTTTGAAGGAAAAAATAACGAATATATTATATGAACAGCAGTGA [SEQ ID NO: 14]MEHNLDMEQLEEIFHSVQHIVWKNSRLIPINFWTFDDYQQEGRLVLYDLLGDGVTQRNLFCHFKVRYKQRLIDIKRRERAFKRGFDCGTGLDIYEYSDALKGKAASPEHILISGSLLEEVFENLNLRYRRLLKSYLAGDELHRMEKYRLKEKITNILYEQQ [SEQ ID NO: 15]ATGGCAGAAAATAATTTAGATAAAGAACAGCTTGAAGAGTTATTCCATTCACTTCAACATATTGTTTGGAAGAACAGTCATTTAATTAAAATAAATTTTTGGACAATGGATGATTATCAGCAAGAAGGGCGACTGGTTTTATACCAGTTACTTGAAGATGGCGTGACACAGGAAAAACTATTTTGCCATTTTAAAGTGCGATATAAGCAACGGTTGATTGATATAAAAAGACGAGAAAGAGCATTTAAGCGGGGTTTTGATTGTGGGGCTGGTTTAGATATATATGAGTATTCTGATGCCCTGAAAGGCAAAGCTACCAGTCCTGAATATAACTTAATTTCAGTTACTTTACTTGAAGAGGTTCATCAAAGTTTGAGTTTGAGATACCGCAATTTATTGGAGAATCATCTGTCAGGAGTGGAGTTGCATCGAATGGAAAAATACCGTTTAAAGGAAAAAATCAAGAGAATACTCTATGAAGAAGAATGA [SEQ ID NO: 16]MAENNLDKEQLEELFHSLQHIVWKNSHLIKINFWTMDDYQQEGRLVLYQLLEDGVTQEKLFCHFKVRYKQRLIDIKRRERAFKRGFDCGAGLDIYEYSDALKGKATSPEYNLISVTLLEEVHQSLSLRYRNLLENHLSGVELHRMEKYRLKEKIKRILYEEE [SEQ ID NO: 17]ATGGAGCATAATTTAGATATGGAGCAGCTGGAAGAGATATTTCATTCTGTTCAACATATTGTATGGAAGAATAGTCGTTTGATTCCGATAAATTTTTGGACGATAGATGACTATCAGCAGGAAGGGCGTTTGGTATTATATGATTTACTTGAGGATGGTGTGACACAAAGAAAACTTTTTTGCCATTTTAAAGTACGTTATAAGCAGAGACTTATTGATATTAAAAGAAGGGAGCGGGCTTTTAAAAGGGGTTTTGACTGTGGGACTGGGCTAGATATTTACGAATATTCAGATGCTTTAAAAGGAAAAGTAGCCAGTCCAGAACATACTCTGATTTCTGGCAGTTTGCTTGAAGAAGTTTTAGAAAACTTAAATTTACGCTACCGTGCTCTTCTTAAAAGTTACCTTGCTGGTGATGAACTGCATCGAATGGAAAAACATCGTTTGAAAGAAAAAATAATAAAAATATTATATGATGAACAGTGA [SEQ ID NO: 18]MEHNLDMEQLEEIFHSVQHIVWKNSRLIPINFWTIDDYQQEGRLVLYDLLEDGVTQRKLFCHFKVRYKQRLIDIKRRERAFKRGFDCGTGLDIYEYSDALKGKVASPEHTLISGSLLEEVLENLNLRYRALLKSYLAGDELHRMEKHRLKEKIIKILYDEQ [SEQ ID NO: 19]TTGAAACCGATCGTTTCAAAATCTATGAGAACATTAAAAATCAATTTTTGGACTACAGAGGATTATCATCAAGAGGGTCTAATTACATTAAATGAAATATTAAATTCAGGATGTAAGGAGTCACAACTATACATTCACTTTAAAGTCAAATATCGACAAAAGCTAATAGACGTGATTAGAAAATCACAGGCGCAAAAAAGAATCTGGGATAATGCAGAGAGTATTGATGTTTACGAATCTGAAAATCAAATTAATTCCAGTAACTCAAACCCCGAAGACATAATAGTCTATGACAGTCTTGTAAAGGAAGTAATAACAAAATTAACACCTTCATACCGGAAACTACTGAAACGACATCTAAGAGGTGAGGATGTGACAAGGATGGAAAAATACAGACTGAAGGAACGAATCAAACAAATTTTATTTGATGGTGATTGA [SEQ ID NO: 20]MKPIVSKSMRTLKINFWTTEDYHQEGLITLNEILNSGCKESQLYIHFKVKYRQKLIDVIRKSQAQKRIWDNAESIDVYESENQINSSNSNPEDIIVYDSLVKEVITKLTPSYRKLLKRHLRGEDVTRMEKYRLKERIKQILFDGD [SEQ ID NO: 21]ATGGATAAGATTGAAACCATACTTAAAAATATTGAACCGATTATCATGAACTGTCGAAAAAAAACTAACATCCCTTCCTGGCAATTAGACGACTATCTCCAGGAAGGCATGATTATTGCTCTAGAGATGTATCATCAACTTTTATTAGACCCACCAGATGATGACTTTAACTTCTATGTTTATTTCAAAGTGAGATATTCTTGTTTCTTGATTGATCAGTATCGGAGAAACATGGCTGTCAAAAGAAAATTCGACCAGATTGACTATTGTGAACTATCTGAGGCGTTTTATCTTTTTGATCAAAATCAAGATGTCTCTGAAAACGTCATGTATAATTTGTTATGTCAAGAAATACACTTGCTTCTATCTCCTGAAGAACGAGAGCTTTTTGAGGCACTTAAAAATGGACAGAAGATTGACCGTAATCAAAAGTTTCGTATCAAGAAGAAAATTATTGAATATATTAAGAGGTTTTGGTGA [SEQ ID NO: 22]MDKIETILKNIEPIIMNCRKKTNIPSWQLDDYLQEGMIIALEMYHQLLLDPPDDDFNFYVYFKVRYSCFLIDQYRRNMAVKRKFDQIDYCELSEAFYLFDQNQDVSENVMYNLLCQEIHLLLSPEERELFEALKNGQKIDRNQKFRIKKKIIEYIKRFW

In some embodiments, said comX gene has the nucleotide sequence of SEQID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or has at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to the nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or is a variant ofSEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 encoding respectively the ComXprotein of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 or is a variant ofSEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 encoding respectively afunctional ComX protein having at least 90% identity or at least 90%similarity to a ComX protein of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.In a particular embodiment, said comX gene is used when the Lactococcusstrain in step a) is a Lactococcus lactis strain.

In a particular embodiment, when the strain of step a) is a Lactococcuslactis subsp. lactis strain, the comX gene comprises the nucleotidesequence of SEQ ID NO:1, any sequence having at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to SEQ ID NO:1, avariant of SEQ ID NO:1 encoding the ComX protein of SEQ ID NO:2 or avariant of SEQ ID NO:1 encoding a functional ComX protein having atleast 90% identity or at least 90% similarity to a ComX protein of SEQID NO:2.

In a particular embodiment, when the strain of step a) is a Lactococcuslactis subsp. cremoris strain, the comX gene comprises the nucleotidesequence of SEQ ID NO:3 or SEQ ID NO:5, any sequence having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identityto SEQ ID NO:3 or SEQ ID NO:5 or a variant of SEQ ID NO:3 or SEQ ID NO:5encoding respectively the ComX protein of SEQ ID NO:4 or SEQ ID NO:6 ora variant of SEQ ID NO:3 or SEQ ID NO:5 encoding respectively afunctional ComX protein having at least 90% identity or at least 90%similarity to a ComX protein of SEQ ID NO:4 or SEQ ID NO:6.

In some embodiments, said comX gene has the nucleotide sequence of SEQID NO:7 or has at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity to the nucleotide sequence of SEQ ID NO:7 or is avariant of SEQ ID NO:7 encoding the ComX protein of SEQ ID NO:8, or is avariant of SEQ ID NO:7 encoding a functional ComX protein having atleast 90% identity or at least 90% similarity to a ComX protein of SEQID NO:8. In a particular embodiment, said comX gene is used when theLactococcus strain in step a) is a Lactococcus raffinolactis strain.

In some embodiments, said comX gene has the nucleotide sequence of SEQID NO:9 or has at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity to the nucleotide sequence of SEQ ID NO:9 or is avariant of SEQ ID NO:9 encoding the ComX protein of SEQ ID NO:10, or isa variant of SEQ ID NO:9 encoding a functional ComX protein having atleast 90% identity or at least 90% similarity to a ComX protein of SEQID NO:10. In a particular embodiment, said comX gene is used when theLactococcus strain in step a) is a Lactococcus plantarum strain.

In some embodiments, said comX gene has the nucleotide sequence of SEQID NO:11 or has at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity to the nucleotide sequence of SEQ ID NO:11 or is avariant of SEQ ID NO:11 encoding the ComX protein of SEQ ID NO:12, or isa variant of SEQ ID NO:11 encoding a functional ComX protein having atleast 90% identity or at least 90% similarity to a ComX protein of SEQID NO:12. In a particular embodiment, said comX gene is used when theLactococcus strain in step a) is a Lactococcus piscium strain.

In a particular embodiment, said comX gene has the nucleotide sequenceof SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17, any sequence having atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or a variant ofSEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 encoding respectively theComX protein of SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or avariant of SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 encodingrespectively a functional ComX protein having at least 90% identity orat least 90% similarity to a ComX protein of SEQ ID NO:14 or SEQ IDNO:16 or SEQ ID NO:18. In a particular embodiment, said comX gene isused when the Lactococcus strain in step a) is a Lactococcus garvieaestrain.

In some embodiments, said comX gene has the nucleotide sequence of SEQID NO:19 or has at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity to the nucleotide sequence of SEQ ID NO:19 or is avariant of SEQ ID NO:19 encoding the ComX protein of SEQ ID NO:20, or isa variant of SEQ ID NO:19 encoding a functional ComX protein having atleast 90% identity or at least 90% similarity to a ComX protein of SEQID NO:20. In a particular embodiment, said comX gene is used when theLactococcus strain in step a) is a Lactococcus fujiensis strain.

In some embodiments, said comX gene has the nucleotide sequence of SEQID NO:21 or has at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity to the nucleotide sequence of SEQ ID NO:21 or is avariant of SEQ ID NO:21 encoding the ComX protein of SEQ ID NO:22, or isa variant of SEQ ID NO:21 encoding a functional ComX protein having atleast 90% identity or at least 90% similarity to a ComX protein of SEQID NO:22. In a particular embodiment, said comX gene is used when theLactococcus strain in step a) is a Lactococcus chungangensis strain.

By way of example and for the avoidance of doubt, in particularembodiments, where a comX gene is specified as having a particularnucleotide sequence, it is understood that the comX gene comprises saidnucleotide sequence. In particular other embodiments, where a comX geneis specified as having a particular nucleotide sequence, it isunderstood that the comX gene consists of said nucleotide sequence.

In some embodiments, variants as defined herein of comX genes areselected from the list of DNA sequences disclosed in Table 1 below:

TABLE 1 Strain Accession number Position of comX, from start to stopLactococcus lactis Al06 CP009472.1 From 2260881 to 2261372 (reverse)Lactococcus lactis Bpl1 JRFX01000055.1 From 34668 to 35159 (forward)Lactococcus lactis Ll1596 LDEK01000015.1 From 33401 to 33892 (forward)Lactococcus lactis subsp. cremoris A17 JQIC01000009.1 From 6445 to 6936(reverse) Lactococcus lactis subsp. cremoris A76 CP003132.1 From 2293232to 2293723 (reverse) Lactococcus lactis subsp. cremoris AM2LITE01000081.1 From 9954 to 10444 (forward) Lactococcus lactis subsp.cremoris B40 LITC01000320.1 From 10186 to 10677 (forward) Lactococcuslactis subsp. cremoris DPC6856 LAVW01000168.1 From 445 to 936 (reverse)Lactococcus lactis subsp. cremoris GE214 AZSI01000020.1 From 186 to 677(reverse) Lactococcus lactis subsp. cremoris HP JAUH01000192.1 From 40to 531 (reverse) Lactococcus lactis subsp. cremoris IBB477JMMZ01000035.1 From 92323 to 92814 (reverse) Lactococcus lactis subsp.cremoris KW10 LIYF01000023.1 From 40421 to 40912 (forward) Lactococcuslactis subsp. cremoris KW2 CP004884.1 From 2276371 to 2276862 (reverse)Lactococcus lactis subsp. cremoris LMG6897 LISZ01000238.1 From 10034 to10525 (forward) Lactococcus lactis subsp. cremoris Mast36 JZUI01000076.1From 310 to 801 (reverse) Lactococcus lactis subsp. cremoris MG1363AM406671.1 From 2376782 to 2377273 (reverse) Lactococcus lactis subsp.cremoris NBRC 100676 BCVK01000073.1 From 9879 to 10370 (forward)Lactococcus lactis subsp. cremoris NZ9000 CP002094.1 From 2377598 to2378089 (reverse) Lactococcus lactis subsp. cremoris SK11 CP000425.1From 2283008 to 2283498 (reverse) Lactococcus lactis subsp. cremorisTIFN1 ASXF01000005.1 From 5621 to 6112 (forward) Lactococcus lactissubsp. cremoris TIFN3 ATBE01000400.1 From 431 to 922 (reverse)Lactococcus lactis subsp. cremoris TIFN5 ATBC01000090.1 From 315 to 809(reverse) Lactococcus lactis subsp. cremoris TIFN6 ATBB01000278.1 From265 to 756 (forward) Lactococcus lactis subsp. cremoris TIFN7ATBA01000081.1 From 5620 to 6111 (forward) Lactococcus lactis subsp.cremoris UC509.9 CP003157.1 From 2107522 to 2108013 (reverse)Lactococcus lactis subsp. cremoris V4 LIYG01000005.1 From 8625 to 9116(forward) Lactococcus lactis subsp. hordniae NBRC 100931 BCVL01000030.1From 70 to 561 (reverse) Lactococcus lactis subsp. lactis 1AA59AZQT01000035.1 From 118 to 609 (reverse) Lactococcus lactis subsp.lactis 511 JNLP01000001.1 From 1703029 to 1703520 (reverse) Lactococcuslactis subsp. lactis A12 LT599049.1 From 2415707 to 2416198 (reverse)Lactococcus lactis subsp. lactis ATCC 19435 LKLC01000004.1 From 32310 to32801 (forward) Lactococcus lactis subsp. lactis bv. diacetylactis DRA4LIWD01000119.1 From 147 to 638 (reverse) Lactococcus lactis subsp.lactis CV56 CP002365.1 From 2213300 to 2213791 (reverse) Lactococcuslactis subsp. lactis DPC6853 LAVD01000101.1 From 544 to 1035 (reverse)Lactococcus lactis subsp. lactis E34 LKLD01000014.1 From 197 to 688(reverse) Lactococcus lactis subsp. lactis Il1403 AE005176.1 From2223528 to 2224019 (reverse) Lactococcus lactis subsp. lactis IO-1 DNAAP012281.1 From 2287126 to 2287617 (reverse) Lactococcus lactis subsp.lactis JCM 7638 BBAP01000017.1 From 34164 to 34656 (forward) Lactococcuslactis subsp. lactis K231 LKLE01000041.1 From 32159 to 32650 (forward)Lactococcus lactis subsp. lactis K337 LKLF01000041.1 From 34909 to 35400(forward) Lactococcus lactis subsp. lactis KF134 LKLJ01000010.1 From34939 to 35430 (forward) Lactococcus lactis subsp. lactis KF147CP001834.1 From 2446402 to 2446893 (reverse) Lactococcus lactis subsp.lactis KF201 LKLM01000024.1 From 28747 to 29238 (forward) Lactococcuslactis subsp. lactis KF24 LKLH01000011.1 From 34116 to 34607 (forward)Lactococcus lactis subsp. lactis KF282 LKLN01000033.1 From 170 to 661(reverse) Lactococcus lactis subsp. lactis KLDS 4.0325 CP006766.1 From2407603 to 2408094 (reverse) Lactococcus lactis subsp. lactis LMG 7760JQCM01000018.1 From 37736 to 38227 (forward) Lactococcus lactis subsp.lactis LMG8526 LKLQ01000046.1 From 38499 to 38993 (forward) Lactococcuslactis subsp. lactis NCDO 2118 CP009054.1 From 2402923 to 2403414(reverse) Lactococcus lactis subsp. lactis S0 CP010050.1 From 2359456 to2359947 (reverse) Lactococcus lactis subsp. lactis UC317 LKLY01000004.1From 36130 to 36621 (forward) Lactococcus lactis WG2 LXWJ01000007.1 From37921 to 38412 (forward) Lactococcus raffinolactis NBRC 100932BCVN01000102.1 From 139 to 617 (forward) Lactococcus piscium CNCM I-4031FLZT01000001.1 From 149 to 628 (forward) Lactococcus piscium MKFS47LN774769.1 From 1708720 to 1709199 (forward) Lactococcus garvieae 122061AP017373.1 From 1356405 to 1356890 (forward) Lactococcus garvieae 8831AFCD01000005.1 From 510 to 995 (forward) Lactococcus garvieaeLg-ilsanpaik-gs201105 JPUJ01000002.1 From 180817 to 181302 (reverse)Lactococcus garvieae LG9 AGQY01000137.1 From 5631 to 6116 (reverse)Lactococcus garvieae M79 FOTJ01000023.1 From 3224 to 3709 (forward)Lactococcus garvieae NBRC 100934 BBJW01000010.1 From 105946 to 106431(reverse) Lactococcus garvieae PAQ102015-99 LXWL01000009.1 From 238437to 238922 (reverse) Lactococcus garvieae TB25 AGQX01000090.1 From 28088to 28573 (reverse) Lactococcus garvieae TRF1 AVFE01000015.1 From 42141to 42626 (reverse)

As used herein a comX gene is understood to be a gene that encodes afunctional ComX protein in the strain where it is expressed. By“functional ComX protein” it is meant a protein which induces or is ableto induce the expression of genes regulated by the Com-box, and at leastone of the late competence genes selected from comFA, comFA, comGA,dprA, coiA, ssbA, radA, radC, recA, and recX.

The ComX protein may have the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22, or anamino acid sequence having at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity to the amino acid sequence of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22,or an amino acid sequence having at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% similarity to the amino acid sequenceof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQID NO:22.

In some embodiments, the ComX protein may have the amino acid sequenceof SEQ ID NO:2, or an amino acid sequence having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to the aminoacid sequence of SEQ ID NO:2 or an amino acid sequence having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%similarity to the amino acid sequence of SEQ ID NO:2. In a particularembodiment, said ComX protein is used when the Lactococcus strain instep a) is a Lactococcus lactis subsp. lactis strain.

In some embodiments, the ComX protein may have the amino acid sequenceof SEQ ID NO:4 or SEQ ID NO:6, or an amino acid sequence having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identityto the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6 or an aminoacid sequence having at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% similarity to the amino acid sequence of SEQ IDNO:4 or SEQ ID NO:6. In a particular embodiment, said ComX protein isused when the Lactococcus strain in step a) is a Lactococcus lactissubsp. cremoris strain.

In some embodiments, the ComX protein may have the amino acid sequenceof SEQ ID NO:8, or an amino acid sequence having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to the aminoacid sequence of SEQ ID NO:8 or an amino acid sequence having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%similarity to the amino acid sequence of SEQ ID NO:8. In a particularembodiment, said ComX protein is used when the Lactococcus strain instep a) is a Lactococcus raffinolactis strain.

In some embodiments, the ComX protein may have the amino acid sequenceof SEQ ID NO:10, or an amino acid sequence having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to the aminoacid sequence of SEQ ID NO:10 or an amino acid sequence having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%similarity to the amino acid sequence of SEQ ID NO:10. In a particularembodiment, said ComX protein is used when the Lactococcus strain instep a) is a Lactococcus plantarum strain.

In some embodiments, the ComX protein may have the amino acid sequenceof SEQ ID NO:12, or an amino acid sequence having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to the aminoacid sequence of SEQ ID NO:12 or an amino acid sequence having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%similarity to the amino acid sequence of SEQ ID NO:12. In a particularembodiment, said ComX protein is used when the Lactococcus strain instep a) is a Lactococcus piscium strain.

In some embodiments, the ComX protein may have the amino acid sequenceof SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18, or an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity to the amino acid sequence of SEQ ID NO:14, SEQ ID NO:16 orSEQ ID NO:18 or an amino acid sequence having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% similarity to the aminoacid sequence of SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18. In aparticular embodiment, said ComX protein is used when the Lactococcusstrain in step a) is a Lactococcus garvieae strain.

In some embodiments, the ComX protein may have the amino acid sequenceof SEQ ID NO:20, or an amino acid sequence having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to the aminoacid sequence of SEQ ID NO:20 or an amino acid sequence having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%similarity to the amino acid sequence of SEQ ID NO:20. In a particularembodiment, said ComX protein is used when the Lactococcus strain instep a) is a Lactococcus fujiensis strain.

In some embodiments, the ComX protein may have the amino acid sequenceof SEQ ID NO:22, or an amino acid sequence having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to the aminoacid sequence of SEQ ID NO:22 or an amino acid sequence having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%similarity to the amino acid sequence of SEQ ID NO:22. In a particularembodiment, said ComX protein is used when the Lactococcus strain instep a) is a Lactococcus chungangensis strain.

According to the invention, when a ComX protein is defined by its aminoacid sequence having a percentage of identity or percentage ofsimilarity to a specific SEQ ID, said ComX protein is a functional ComXprotein as defined herein.

By way of example and for the avoidance of doubt, in particularembodiments, where a ComX protein is specified as having a particularamino acid sequence, it is understood that the ComX protein comprisessaid amino acid sequence. In particular other embodiments, where a ComXprotein is specified as having a particular amino acid sequence, it isunderstood that the ComX protein consists of said amino acid sequence.

In some embodiments, ComX proteins having percentage of identity orpercentage of similarity as defined herein are selected from the list ofprotein sequences derived, after translation, from the list of DNAsequences disclosed in Table 1 above.

Preferably, reference to a sequence which has a percentage identity orsimilarity to any one of the SEQ ID NOs detailed herein refers to asequence which has the stated percent identity or similarity with theSEQ ID NO referred to, over the entire length of the two sequences.Percentage (%) sequence identity is defined as the percentage of aminoacids or nucleotides in a candidate sequence that are identical to theamino acids or nucleotides in a reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Percentage (%) sequence similarity is definedas the percentage of amino acids in a candidate sequence that aresimilar to the amino acids in a reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence similarity. Similarity between amino acids is based onestablished amino acid substitution matrices such as the PAM series(Point Accepted Mutation; e.g. PAM30, PAM70, and PAM250) or the BLOSUMseries (BLOck SUbstitution Matrix; e.g. BLOSUM45, BLOSUM50, BLOSUM62,BLOSUM80, and BLOSUM90). Alignment for purposes of determining percentsequence identity or similarity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as CLUSTALW, CLUSTALX, CLUSTAL Omega, BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. In a particularembodiment, similarity between amino acids is determined using theBLASTp software with the BLOSUM62 matrix. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared, or gappenalties to be introduced, can be determined by known methods.

In a particular embodiment, when the modulation in step b) results froman exogenous comX gene, said exogenous comX gene is (obtained) from astrain of the same species, in particular of the same subspecies, as thestrain provided in step a). In any case, the exogenous comX gene needsto be functional, in particular needs to encode a functional ComXprotein, as defined herein in the strain provided in step a).

Exogenous DNA Polynucleotide

The method of the present invention comprises the step of contacting thestrain of step b) with an exogenous DNA polynucleotide in a medium andincubating the resulting mixture for integration of the exogenous DNAinto the genome of said strain [step c].

As used herein the term “exogenous DNA polynucleotide” refers to a DNApolynucleotide that is brought into the cytoplasm of said strain, inorder to be integrated into the genome of said strain (target sequence).

In a particular embodiment, the method comprises carrying out step (b)[ComX modulation] and then carrying out step (c) [contact with theexogenous DNA polynucleotide] [i.e., that step (c) is carried out on astrain obtained following step (b)]. Thus, the method comprising thesteps of:

-   -   (a) providing a strain of the Lactococcus genus, wherein said        strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain obtained in step (b) with an        exogenous DNA polynucleotide in a medium and incubating the        resulting mixture for integration of the exogenous DNA        polynucleotide into the genome of said strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome.

In another embodiment, the method comprises carrying out simultaneouslystep (b) [ComX modulation] and step (c) [contact with the exogenous DNApolynucleotide]. This option is appropriate when the ComX modulation isthe result of the expression of the endogenous comX gene or of theincrease of the expression of the endogenous comX gene of said strain.Thus, the method comprising the steps of:

-   -   (a) providing a strain of the Lactococcus genus, wherein said        strain is transformable through natural competence;    -   (b) modulating the production of a ComX protein in said strain;    -   (c) contacting said strain with an exogenous DNA polynucleotide        in a medium and incubating the resulting mixture for integration        of the exogenous DNA polynucleotide into the genome of said        strain; and    -   (d) selecting a strain which has integrated the exogenous DNA        polynucleotide into its genome;    -   wherein step (b) and step (c) are carried out simultaneously.

In a particular embodiment, the sequence of the exogenous DNApolynucleotide used in step c) share some similarities or identitieswith the genome of the Lactococcus strain to be transformed (of step a).In a particular embodiment, the exogenous DNA polynucleotide used instep c) is designed such that its 5′ part and its 3′ part are identicalor highly similar to parts of the genome of the Lactococcus strain to betransformed (of step a), while its central part can be different fromthe genome of the Lactococcus strain to be transformed (of step a). Thehigh similarity of the arms with the regions surrounding the targetsequence can be determined by the person skilled in the art using commongeneral knowledge, in particular by reference to homologousrecombination.

Thus, to replace a target sequence by a mutated sequence or a truncatedsequence or a supplementary sequence in the genome of the Lactococcusstrain to be transformed (of step a), the exogenous DNA polynucleotideused in step c) is designed such that:

-   -   its 5′ part is identical or highly similar to the region of the        genome of the Lactococcus strain to be transformed which is on        one side of the target sequence;    -   its central part contains the replacing sequence (i.e., the        mutated sequence or the truncated sequence or the supplementary        sequence); and    -   its 3′ part is identical or highly similar to the region of the        genome of the Lactococcus strain to be transformed which is on        the other side of the target sequence.

The 5′ part and 3′ part are long enough to ensure efficientrecombination. In a particular embodiment, each of the 5′ part and 3′part is from 0.5 to 5 kb in length. The size of the arms can bedetermined by the person skilled in the art using common generalknowledge, in particular by reference to homologous recombination.

In a particular embodiment, the exogenous DNA polynucleotide used instep c) is (obtained) from a strain of the Lactococcus genus.

In a particular embodiment, said exogenous DNA polynucleotide used instep (c) is (obtained) from a strain of the same species, in particularof the same subspecies, as the strain provided in step (a).

In a particular embodiment, the exogenous DNA polynucleotide used instep c) is from a strain of the Lactococcus lactis species. In aparticular embodiment, the exogenous DNA polynucleotide used in step c)is from a strain of the same Lactococcus lactis subspecies as the strainprovided in step a). In a particular embodiment, the exogenous DNApolynucleotide used in step c) is from a strain of a Lactococcus lactissubspecies which is different from the strain provided in step a).

In a particular embodiment, the exogenous DNA polynucleotide used instep c) is from a strain of the Lactococcus raffinolactis species

The exogenous DNA polynucleotide may encode part of a gene sequence, agene sequence, or a plurality of gene sequences. The gene sequence maybe operably linked to transcription regulator(s). In a particularembodiment, the exogenous DNA polynucleotide is linear. The exogenousDNA polynucleotide may be designed to facilitate its incorporationwithin the genome of the L. lactis strain by homologous recombination(e.g. the exogenous DNA polynucleotide may comprise one or morerecombination arms). The exogenous DNA polynucleotide may be a singlestranded linear DNA.

The exogenous DNA polynucleotide, when incorporated into the genome ofsaid Lactococcus strain leads to genetic modification of the strain suchas gene replacement (to add or to remove a mutation), gene addition (toadd a new gene or to duplicate an existing gene), gene deletion (toremove part or the totality of a gene), modification of non-codingregion (to modulate expression of a gene). Typically, the exogenous DNApolynucleotide, when incorporated into the genome of said Lactococcusstrain confers an interesting or useful phenotype, e.g. modified kineticof acidification, improved resistance to bacteriophage, modifiedcapability to grow in milk, modified texturing properties, improvedsafety of the strain. For example, improved bacteriophage resistancecould be achieved by incorporating genes coding for arestriction/modification system into the strain genome or by introducinga mutation or a deletion into the pip gene.

As an example, growth of a L. lactis strain in milk could be improved byinserting into the chromosome the prtP and prtM genes that allow caseinhydrolysis and better nitrogen nutrition; alternatively, these genescould be inactivated to reduced milk proteolysis in cheese. hisDC andtyrDC are genes known to be responsible for biogenic amine production(histamine and tyramine, respectively) in a diversity of lactic acidbacteria; disruption or mutation of these genes could help to preventsafety issues related to cheese consumption.

In a particular embodiment, the exogenous DNA polynucleotide has aminimal size selected from the group consisting of 100 bp, 200 bp, 500bp, 1 kb, 2 kb and 5 kb, and a maximal size selected from the groupconsisting of 500 bp, 1 kb, 2 kb, 5 kb, 10 kb, 20 kb and 50 kb. In aparticular embodiment, the size of the exogenous DNA polynucleotide maybe between 100 bp and 50 kb, more preferably between 500 bp to 20 kb,even more preferably between 1 kb to 10 kb.

The concentration of exogenous DNA polynucleotide in the medium of step(c) may be between 0.5 mg/L and 1 g/L, preferably between 1 mg/L and 500mg/L, more preferably between 5 mg/L and 100 mg/L, even more preferablybetween 10 mg/L and 50 mg/L of medium.

Selection of Transformed Strains

The method of the present invention comprises the step of selecting astrain which has integrated the exogenous DNA polynucleotide into itsgenome [step d)].

If needed, selection is carried out on some cells of colonies that havebeen previously obtained by multiplying, in the appropriate medium,cells obtained at the end of step c) (or at the end of the simultaneoussteps b) and c), when appropriate).

Various methods for the selection of transformed bacteria are well knownin the art (see, e.g. Sambrook et al.) and may be routinely applied bythe person skilled in the art, such as PCR, DNA sequencing . . . .

For example, when the exogenous DNA polynucleotide used in step c)provides a particular phenotype that the Lactococcus strain of step a)does not display (either a new phenotype or restoring a lost phenotype),it is possible to select strains which have integrated the exogenous DNApolynucleotide into their genome by selecting strains expressing thephenotype. This is the case for a strain having integrated in its genomean exogenous DNA polynucleotide mutated for the pip gene (that providesresistance to some bacteriophages).

For example, when the exogenous DNA polynucleotide used in step c) leadsonce integrated to a loss of a phenotype initially displayed by theLactococcus strain of step a), it is possible to select strains whichhave integrated the exogenous DNA polynucleotide into their genome byselecting strains which do not display the phenotype any more. This isthe case for an exogenous DNA polynucleotide bearing a mutated hisDC ortyrDC gene, which suppresses or decreases the production of histamine ortyramine, respectively.

As a particular example, the exogenous DNA polynucleotide may bear anantibiotic resistance gene. Accordingly, a Lactococcus strain which hasintegrated the exogenous DNA polynucleotide into its genome may beselected by plating onto a medium comprising said antibiotic. Onlystrains that express the appropriate antibiotic resistance gene, as aresult of a successful transformation with the exogenous DNApolynucleotide, will multiply.

Growth Conditions

As described in Example 3, a positive effect on natural competenceinduction in L. lactis strains was observed when cells were pre-culturedin a complex medium before transferring the cells to a chemicallydefined medium (FIG. 3).

Accordingly, in some embodiments the medium of step (c) is a chemicallydefined medium. As used herein, the term “chemically defined medium”(CDM) refers to a medium for which the exact chemical composition isknown. Preferably, the CDM may have the composition of the CDM set outin Sissler et al. (1999, Proc Natl Acad Sci USA 96:8985-8990). Thus, inan embodiment, the chemically defined medium (CDM) comprises 0.5 g/LNH₄Cl, 9.0 g/L KH₂PO₄, 7.5 g/L K₂HPO₄, 0.2 g/L MgCl₂, 5 mg/L FeCl₂, 50mg/L CaCl₂), 5 mg/L ZnSO₄, 2.5 mg/L CoCl₂, 0.05 g/L tyrosine, 0.1 g/Lasparagine, 0.1 g/L cysteine, 0.1 g/L glutamine, 0.1 g/L isoleucine, 0.1g/L leucine, 0.1 g/L methionine, 0.1 g/L tryptophan, 0.1 g/L valine, 0.1g/L histidine, 0.2 g/L arginine, 0.2 g/L glycine, 0.2 g/L lysine, 0.2g/L phenylalanine, 0.2 g/L threonine, 0.3 g/L alanine, 0.3 g/L proline,0.3 g/L serine, 10 mg/L paraaminobenzoic acid, 10 mg/L biotin, 1 mg/Lfolic acid, 1 mg/L nicotinic acid, 1 mg/L panthotenic acid, 1 mg/Lriboflavin, 1 mg/L thiamine, 2 mg/L pyridoxine, 1 mg/L cyanocobalamin, 5mg/L orotic acid, 5 mg/L 2-deoxythymidine, 5 mg/L inosine, 2.5 mg/Ldl-6,8-thioctic acid, 5 mg/L pyridoxamine, 10 mg/L adenine, 10 mg/Lguanine, 10 mg/L uracil, 10 mg/L xanthine, and 5 g/L glucose..

In some embodiments, prior to step (c) said strain is incubated in apre-culture medium, preferably wherein the pre-culture medium is acomplex medium, more preferably wherein the pre-culture medium is M17G(i.e., the M17 medium supplemented with glucose) or THBG (i.e., the THBmedium supplemented with glucose).

The complex medium may be Todd Hewitt broth (THB) (Todd and Hewitt,1932; Updyke and Nickle, 1954) or M17 broth (Terzaghi and Sandine,1975). THB may comprise 500 g/L beef heart infusion, 20 g/L pepticdigest of animal tissue, 2 g/L dextrose, 2 g/L sodium chloride, 0.4 g/Lsodium phosphate, 2.5 g/L sodium carbonate. M17 broth may comprise: 0.5g/L ascorbic acid, 5 g/L lactose, 0.25 g/L magnesium sulfate, 5 g/L meatextract, 2.5 g/L meat peptone (peptic), 19 g/L sodium glycerophosphate,5 g/L soya peptone (papainic), 2.5 g/L tryptone, 2.5 g/L yeast extract.

Method for Identifying Strains Transformable by Natural Competence

In another aspect, the present invention relates to a method foridentifying a strain of the Lactococcus genus which is transformablethrough natural competence. Said method comprises the following steps:

-   -   (a) providing a strain of the Lactococcus genus;    -   (b) transforming said strain with a plasmid expressing a comX        gene having at least 90% identity, preferably having 100%        identity, to the endogenous comX gene of said strain;    -   (c) contacting said strain obtained in step (b) with an        exogenous marker DNA polynucleotide in a medium and incubating        the resulting mixture for integration of the exogenous DNA        polynucleotide into the genome of said strain; and    -   (d) determining the rate of recombination events;

wherein a rate of at least 1×10⁻⁶ transformants per μg of DNA isindicative of a strain which is transformable through naturalcompetence.

The term “rate of recombination events” may be used interchangeably withthe term “transformation rate”. The rate of recombination events iscalculated by determining the ratio of the number of cells havingintegrated the exogenous marker DNA polynucleotide over the total numberof viable cells. A rate of at least 10⁻⁶ was selected as a threshold,based on the observation that the level of spontaneous mutation inlactococci is less than 10⁻⁶, typically around 10⁻⁷ mutants per μg ofDNA [spontaneous means with no comX expression or overexpression].

By “at least 90% identity to the endogenous comX gene of said strain”,it is meant—as particular embodiments of the method—at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity. In a particularembodiment, said comX gene has 100% identity to the endogenous comX geneof said strain.

In a particular embodiment, said method is implemented with a strain ofthe Lactococcus genus selected from the group consisting of Lactococcuslactis, Lactococcus raffinolactis, Lactococcus plantarum, Lactococcuspiscium, Lactococcus garivieae, Lactococcus fujiensis and Lactococcuschungangensis.

In a particular embodiment, said method is implemented with a strain ofthe Lactococcus lactis species. Said method comprises the followingsteps:

-   -   (a) providing a strain of the Lactococcus lactis species;    -   (b) transforming said strain with a plasmid expressing a comX        gene having at least 90% identity to the polynucleotide sequence        of SEQ ID NO:1, 3 or 5;    -   (c) contacting said strain obtained in step (b) with an        exogenous marker DNA polynucleotide in a medium and incubating        the resulting mixture for integration of the exogenous DNA        polynucleotide into the genome of said strain; and    -   (d) determining the rate of recombination events;

wherein a rate of at least 1×10⁻⁶ transformants per μg of DNA isindicative of a strain of the Lactococcus lactis species which istransformable through natural competence.

In a particular embodiment, said method is implemented with a strain ofthe Lactococcus raffinolactis species. Said method comprises thefollowing steps:

-   -   (a) providing a strain of the Lactococcus raffinolactis species;    -   (b) transforming said strain with a plasmid expressing a comX        gene having at least 90% identity to the polynucleotide sequence        of SEQ ID NO:7;    -   (c) contacting said strain obtained in step (b) with an        exogenous marker DNA polynucleotide in a medium and incubating        the resulting mixture for integration of the exogenous DNA        polynucleotide into the genome of said strain; and    -   (d) determining the rate of recombination events;    -   wherein a rate of at least 1×10⁻⁶ transformants per μg of DNA is        indicative of a strain of the Lactococcus lactis species which        is transformable through natural competence.

In some embodiments, the comX gene is from a strain of the same species,in particular of the same subspecies, as the strain provided in step a).In some embodiments, the comX gene is identical (100% identity) to thepolynucleotide sequence of the endogenous comX gene of the strain ofstep a).

In some embodiments, when the strain of step a) is a Lactococcus lactissubsp. lactis strain, the comX gene has at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to thepolynucleotide sequence of SEQ ID NO:1.

In some embodiments, when the strain of step a) is a Lactococcus lactissubsp. cremoris strain the comX gene has at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to thepolynucleotide sequence of SEQ ID NO:3 or SEQ ID NO:5.

In some embodiments, when the strain of step a) is a Lactococcusraffinolactis strain the comX gene has at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to thepolynucleotide sequence of SEQ ID NO:7.

In some embodiments, when the strain of step a) is a Lactococcusplantarum strain the comX gene has at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to the polynucleotidesequence of SEQ ID NO:9.

In some embodiments, when the strain of step a) is a Lactococcus pisciumstrain the comX gene has at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identity to the polynucleotide sequenceof SEQ ID NO:11.

In some embodiments, when the strain of step a) is a Lactococcusgarvieae strain the comX gene has at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to the polynucleotidesequence of SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17.

In some embodiments, when the strain of step a) is a Lactococcusfujiensis strain the comX gene has at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to the polynucleotidesequence of SEQ ID NO:19.

In some embodiments, when the strain of step a) is a Lactococcuschungangensis strain the comX gene has at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to thepolynucleotide sequence of SEQ ID NO:21.

It is preferable to use, as an exogenous marker DNA polynucleotide, apolynucleotide bearing a gene which is initially not present in theLactococcus strain of step a) [even as a mutated version]. This wouldavoid that during step c) the Lactococcus strain acquires a functionalgene by other means than natural competence, e.g. by spontaneousmutation of its genome.

As an example, the exogenous marker DNA polynucleotide bears a geneencoding a luciferase gene. Accordingly, a Lactococcus strain which hasintegrated the exogenous DNA polynucleotide into its genome may beselected for expression of the luciferase. Only strains that express theluciferase gene (i.e., integrated) will be detectable bybioluminescence.

As another example, the exogenous marker DNA polynucleotide bears anantibiotic resistance gene. Accordingly, a Lactococcus strain which hasintegrated the exogenous DNA polynucleotide into its genome may beselected by plating the cells onto a medium comprising said antibiotic.

An example of a method for identifying a strain of the Lactococcus genuswhich is transformable through natural competence according to thepresent invention (Assay A) may be performed using the following steps:

-   -   i) Providing a strain of the Lactococcus genus, in particular of        the Lactococcus lactis species.    -   ii) Transforming said strain with a plasmid expressing a comX        gene having at least 90% identity, preferably having 100%        identity to the endogenous comX gene of said strain (e.g. the        pGhP32comX_(MG) plasmid of Materials and Methods).    -   iii) Pre-culturing the transformed strain overnight in a complex        medium supplemented with glucose (e.g. M17G) at 30° C.    -   iv) Diluting about 1.5 mL of the pre-culture in about 8.5 mL of        fresh medium.    -   v) After about 2 hours further growth at 30° C., washing the        cells twice with distilled water and adjusting the OD₆₀₀ to 0.05        in a chemically defined medium (e.g. CDM) containing 5 μg mL⁻¹        erythromycin and an osmo-stabilizer (e.g. 5% [v/v] glycerol or        5% [w/v] mannitol).    -   vi) Adding 5 μg of exogenous DNA polynucleotide bearing an        antibiotic resistance gene to 300 μl of the culture medium (e.g.        the 3.7 kb PCR product generated from the pGEMrpsL plasmid as        described in Materials and Methods).    -   vii) Incubating the resulting culture for about 6 hours at 30°        C.    -   viii) Plating the cells onto agar plates comprising the complex        medium supplemented with glucose (e.g. M17G) and appropriate        antibiotic (i.e. corresponding to the antibiotic resistance gene        of the exogenous DNA polynucleotide) and incubating for about 48        hours.    -   ix) Counting the colony forming units (CFU) and determining the        transformation rate, wherein a transformation rate of at least        1×10⁻⁶ transformants per μg of DNA is indicative of a strain        which is transformable through natural competence.

The transformation rate may be calculated as the number ofantibiotic-resistance CFU mL⁻¹ divided by the total number of viable CFUmL⁻¹.

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting examples.

EXAMPLES Example 1: Induction of the comGA Promoter by Constitutive comXExpression in Various Strains of the Lactococcus Species

a) In Lactococcus lactis Subsp. Cremoris Strains (MG1363 and KW2)

To test the ability of ComX to induce the late competence genes inLactococcus lactis subsp. cremoris strains, a constitutive comXexpression plasmid (pGhP32comX_(MG)) was created by cloning the comXgene from strain MG1363, under the control of the lactococcal P₃₂promoter on the thermosensitive plasmid pG⁺host9. The latter wasintroduced in strain KW2 that contains a chromosomally-encodedP_(comGA[MG])-luxAB transcriptional fusion (BLD101). The promoter of thelate competence gene comGA (P_(comGA)) contains a putative ComX-bindingmotif and is used here as proxy for competence activation in the ComXstrain.

As alternative for more resistant strains to electro-transformation, andsubsequently to chromosome integration, a portable luminescent reportersystem was also constructed. This replicative plasmid carries theluminescent reporter P_(comGA[MG])-luc with the P₃₂-comX_(MG) cassette.The pGhP32comX_(MG)-P_(comGA[MG])-luc plasmid was transformed in strainMG1363. Specific P_(comGA[MG])-luc/luxAB activities were monitored forthe different strains constructed. The luminescent assays were performedin rich (M17G) and/or CDM media comparing the luciferase activitybetween the overexpressing comX strain and its related negative control(no additional comX copy).

In the KW2 strain containing the P_(comGA[MG])-luxAB reporter asmono-copy in their chromosome, specific luciferase activity was observedfor KW2 containing the P₃₂-comX cassette allowing the constitutiveproduction of ComX. This confirms that comX expression can be carriedout in various L. lactis subsp. cremoris strains using an exogenous comXgene obtained from the same strain or from a strain of the samesubspecies. Eight recombinant clones of the KW2 ComX⁺ reporter strainwere randomly selected and their specific luciferase activity wasmonitored in CDM growth conditions. This medium was chosen because itwas shown to be permissive for competence development in variousstreptococcal species. To ensure reproducibility of the assay,exponentially-growing cells in complex medium (M17 conditions) werewashed and inoculated in fresh CDM before starting the experiment. Asexpected, all tested ComX⁺ clones (cl01 to cl08) displayed between 10¹-and 10⁴-fold higher specific luciferase (Lux) activity than the controlstrain carrying the empty vector (FIGS. 2A and 2B).

Similar results were obtained with the portable luminescent reportersystems in MG1363 (FIG. 2C).

These results strongly suggest that, in the L. lactis subsp. cremorisstrains MG1363 and KW2, ComX induces the comG operon. Additionally,these observations validate these reporter fusions (both chromosomal andplasmid-borne) as a tool to identify conditions capable to activate thecomG-operon which is essential to natural transformation.

b) In a L. lactis Subsp. Lactis Strain (IL1403)

A constitutive comX expression plasmid (pGhP32comX_(IO)) was created bycloning the comX gene from strain IO-1, under the control of thelactococcal P₃₂ promoter on the thermosensitive plasmid pG⁺host9. Aportable luminescent reporter system was also constructed; thisreplicative plasmid carries the luminescent reporter P_(comGA[IO])-lucwith the P₃₂-comX_(IO) cassette. The promoter of the late competencegene comGA (P_(comGA)) contains a putative ComX-binding motif and isused here as proxy for competence activation in the ComX⁺ strain.

This replicative plasmid pGhP32comX_(IO)-P_(comGA[IO])-luc wastransformed in strain IL1403 and specific P_(comGA[IO])-luc activitieswere monitored. One of the IL1403 transformants produced specificP_(comGA[IO])-luc activities confirming that ComX induces the comGoperon (FIG. 2D).

Example 2: Analysis of Essential Late Corn Genes Present in L. lactisGenomes

Among L. lactis strains, genomic variability was previously investigatedfor comX and dprA alleles (Wydau et al., 2006). While all strains(31/31) display a complete version of comX, the dprA content is variableamong subspecies: 50% of the lactis strains (10/20) contain nonsensemutations in dprA while all cremoris strains (11/11) harbor an intactand potentially functional dprA gene.

Since dprA is hypothesized to be important in the natural competencemechanism, its integrity in L. lactis strains prompted us to furtheranalyze the minimal set of late corn genes (17 candidate genes includingcomX; FIG. 1) in the genomes of 3 subsp. cremoris strains and 1 subsp.lactis strain which are publicly available (strains MG1363, SK11, KW2and IL1403). This in silico analysis reveals that the genome of SK11contains a high number of pseudogenes in key competence genes (between 5and 8 incomplete late corn genes) due to transposon insertion orframeshifting events (nucleotide(s) insertion or deletion). Inparticular, the presence of transposable elements in comGA and/or comECgenes, which are respectively essential for pilus assembly and DNAtransport, strongly suggests that natural transformation is no morefunctional in those strains. Although the set of full-length competencegenes in the laboratory strain MG1363 is larger, mutations in comEC(nucleotide insertion) and coiA (nonsense mutation) probably impair itsability to transform DNA by competence (Wegmann et al., 2007). Thosemutations were also found in the genome of its isogenic derivativeNZ9000, which strongly suggests that they do not result from DNAsequencing errors. As far as the L. lactis subsp. lactis IL1403 strainis concerned, its dprA gene contains nonsense mutations probablyimpairing its ability to transform DNA by competence. In contrast,strain KW2 of plant origin (corn fermentation) contains the whole set ofknown essential late genes required to fulfil natural DNAtransformation, making it the best candidate to further study thefunctionality of competence in the cremoris subspecies. Two otherstrains from our collection, L. lactis subsp. lactis SL12651 andSL12653, were also found to contain the whole set of known essentiallate genes (FIG. 1).

Example 3: Effect of Growth Conditions on ComX Activation

We investigated the effect of pre-culturing and culturing conditions(M17G, THBG, and CDM) on the activation of the reporter fusion in theComX⁺ strain. For this purpose, clone 02 (FIG. 2A) was selected since itexhibits the strongest Lux activity. Interestingly, more than a 20-foldvariation in the maximum Lux activity was dependent on the pre- andculturing medium which was used (FIG. 3). Particularly, a positiveimpact of the transition of pre-culture cells from a complex medium to adefined medium was observed. The highest specific Lux activity (˜3×10⁶RLU OD₆₀₀ ⁻¹) was obtained for a switch from M17G to CDM, followed byTHBG to CDM, while all other combinations gave lower activities (between˜1.5× and 5.5×10⁵ RLU OD₆₀₀ ⁻¹). This indicates that first a chemicallydefined medium is superior for maximizing activation of late corn genesof L. lactis KW2 than complex rich media, but also that the switch fromcomplex medium (e.g. M17G or THBG) to defined medium is critical.

Together, these results show that ComX is functional in strain KW2 whenit is constitutively produced (i.e. expressed) and that growthconditions have a significant impact on the activation of late corngenes.

Example 4: Constitutive comX Expression Induces Natural Transformation

a) Acquisition of Single Mutations in the KW2 Genome from Exogenous DNA

We first tested the transfer of single point mutations in the chromosomeof the ComX⁺ KW2 strain. The transforming PCR fragments used encompassthe mutated rpsL allele of a spontaneous streptomycin-resistant(Str^(r)) clone of L. lactis subsp. cremoris MG1363 (strA1 allele, alsocalled rpsL*). This mutated allele bears an A→T substitution at position167 [resulting in the altered ribosomal protein S12 with mutation K56I]as compared to the sequence of the wild-type, streptomycin-sensitiveMG1363. In addition to this mutation, the two rpsL alleles differ by asilent nucleotide substitution at position 39 (T→G). The sequence of therpsL (wild-type) and rpsL* (conferring streptomycin resistance) allelesare disclosed respectively as SEQ ID NO:23 and NO:24 (FIG. 4A).Independently of these two substitutions located at positions 39 and167, the rpsL alleles of KW2 and MG1363 differ by a nucleotidesubstitution at position 156 (A in MG1363, T in KW2). The rpsL allele ofKW2 is disclosed as SEQ ID NO:25 (FIG. 4A). To ensure efficientrecombination, the transforming PCR product also contains upstream anddownstream recombination arms of ˜1.85 kb surrounding the strA1mutation. Transformation assays were performed with the eight previouslyselected clones of the ComX⁺ reporter strain (BLD101 [pGhP32comX_(MG)])and the control strain (BLD101 [pG⁺host9], empty vector) using thestandard protocol reported in Material and Methods. Validation ofnatural transformation is made by sequencing the rpsL region coveringthe point mutations from the donor DNA conferring streptomycinresistance using primers RpsL Univ UP and RpsL Univ DN.

Remarkably, the ComX⁺ clones 02 and 04 that displayed the highestP_(comGA) activation (≥7×10⁵ RLU OD₆₀₀ ⁻¹) yielded mutation frequencies˜15-fold higher than the background level of spontaneous mutation thatwas calculated in the absence of DNA (FIG. 4B). After subtraction of thebackground, a transformation rate of up to 4×10⁻⁵ transformants per μgof DNA (˜10⁴ transformants ml⁻¹) was obtained for clone 02 whichdisplays the highest P_(comGA) activation. In contrast, the negativecontrol strain had a spontaneous mutation rate of ˜1×10⁻⁷ transformantsper μg of DNA.

The rpsL ORF of 10 Str^(r)-derivatives of cl02 was amplified by PCR andsequenced. In all cases, we observed the co-transfer of strA1 (mutationA→T at position 167 of the rpsL gene) and the closely-located T→Amutation at position 156. In some cases, the T→G mutation at position 39was also co-transferred with strA1. The chimeric nature of rpsL in someStr^(r) ComX⁺ derivatives of KW2 (i.e. presence of both mutations atpositions 156 and 167 without the mutation at position 39) ultimatelydemonstrates that a recombination process occurred between the exogenousand chromosomal DNA (FIG. 4A). In contrast, this rearrangement was notobserved in the rpsL gene of spontaneous Str^(r) mutants obtained in thenegative control experiments (i.e. assays performed in absence ofexogenous DNA, or with the control strain carrying the empty vector inpresence of exogenous DNA). These results show that exogenous DNA canenter KW2 cells and be integrated in their chromosome by homologousrecombination when a certain threshold of comX expression is reached.

b) Construction of Deletion Mutants by Natural Competence in L. lactisSubsp. Cremoris KW2 Overexpressing comX

The previous result (Example 4, section a) strongly suggests that DNAtransfer occurs in L. lactis KW2. The 3 mutations transferred by naturaltransformation are grouped on a 128-bp fragment. If a longer DNAfragment could be similarly integrated in the L. lactis chromosomeremains to be determined.

We wondered if overlap PCR as donor DNA could equivalently allow geneinsertions or gene deletions. The idea was to replace the target gene byan antibiotic resistance cassette, i.e. the chloramphenicol resistancecassette P₃₂-cat. For this purpose, a DNA fragment was constructed byoverlap PCR containing the P₃₂-cat cassette flanked by two homologousarms (minimum ˜1.5 kb) containing the upstream and downstream regions ofthe targeted gene.

To this end, exogenous DNA polynucleotides containing P₃₂-cat surroundedby KW2-specific recombination arms (˜1.5 kb) were assembled in vitro byoverlapping PCR to target the comEC, mecA, ciaRH, covRS or clpC gene(see Materials and Methods for details) and transferred by naturaltransformation in the ComX⁺ strain (cl02). Validation of naturaltransformation is made by sequencing the targeted region (comEC, mecA,ciaRH, covRS or clpC, which should contain the chloramphenicolresistance cassette P₃₂-cat) using primers listed in Table 3.

The transformation rate observed for overlap PCR products was ˜1.2×10⁻⁶to 1.1×10⁻⁴ transformants per μg of DNA for the different overlap DNAfragments that were tested (see FIG. 5). Compared to the transformationrate observed for the exchange of a homologous DNA fragment containingonly three point mutations (rpsL* donor DNA; 8×10⁻⁴ transformants per μgof DNA), these rates are relatively high for DNA double recombinationdeletion/replacement.

Example 5: A KW2 ΔcomEC Mutant is Unable of Natural CompetenceTransformation

To confirm that the observed horizontal DNA transfer in ComX⁺ KW2 cellswas indeed mediated by natural competence, and not by phage transductionor conjugation, we investigated the role of the ComEC protein, which isessential for the uptake of transforming DNA through the cell membrane(the comFA gene, together with the comFA, comGA, dprA, coiA, ssbA, radA,radC, recA and recX genes are preceded by a Com-box and have been foundto be activated in KW2 following constitutive comX expression; data notshown).

To create the ΔcomEC strain, clone 02 of the ComX⁺ reporter strain,which was tested above, was grown in CDM conditions in presence of PCRproducts encompassing the comEC gene disrupted by the insertion of thechloramphenicol resistance cassette P₃₂-cat (see Materials and Methods).Four mutants with disrupted comEC (BLD102 [pGhP32comX_(MG)] cl01 tocl04) were validated by PCR for P₃₂-cat insertion in comEC.Transformation assays with the mutated rpsL allele showed that thefrequencies of appearance for Str^(r) clones in all tested ΔcomECderivatives were similar to the background level of spontaneous rpsLmutation frequencies (<10⁻⁷) (FIG. 6). Although heterogeneity inP_(comGA) activation was observed between clones as previously reportedfor the WT ComX⁺ reporter strain, half of the ΔcomEC derivative clones(i.e. cl01 and cl03) displayed maximum specific Lux activity similar tothe transformable WT strains (>1.0×10⁶ RLU OD₆₀₀ ⁻¹) (FIG. 4B). Thisshows that the transformation defect in these ΔcomEC clones does notresult from a too low production of ComX.

Taken together, these results demonstrate that natural DNAtransformation could be activated by ComX overexpression in L. lactissubsp. lactis KW2. Moreover, to the best of our knowledge, these dataprovide the first ever experimental evidence of transformation of L.lactis by natural competence.

Example 6: Natural Competence in Two Strains of the L. RaffinolactisSpecies

Following the positive results obtained regarding natural competence inLactococcus lactis strains, other strains of the Lactococcus genus weretested. Two strains of L. raffinolactis were able to capture plasmidpGhost-Core (15 μg/300 μl) used as donor DNA: LMG13098 and LMG14164.These results suggest that these two strains of L. raffinolactis arenaturally competent for plasmid transformation and that, in thesestrains, natural competence is independent of artificialcomX-overexpression.

The fact that another Lactococcus species could be transformed bycompetence opens additional possibilities for industrial applications.

Example 7: Transformation by Natural Competence in 2 Lactococcus lactisSubsp. Lactis Strains

Two Lactococcus lactis subsp. lactis strains, SL12651 and SL12653,carrying all the essential late corn genes (FIG. 1) were tested. Asdonor DNA, PCR fragments which encompass the mutated rpsL allele (rpsL*)of a spontaneous streptomycin-resistant (Str^(r)) clone of L. lactissubsp. lactis IL1403 was used. Cells were pre-cultured overnight in acomplex medium supplemented with glucose (e.g. M17G) at 30° C. Cellswere washed twice with distilled water and inoculated at an OD₆₀₀ of0.05 in 200 μl M17G containing 25 μg mL⁻¹ donor DNA rpsL*. After 24hours of culture at 30° C., cells incubated or not with donor DNA werespread onto agar plates comprising the complex medium supplemented withglucose (e.g. M17G) and appropriate antibiotic (i.e. streptomycin). CFUswere counted after 48 hours of incubation at 30° C. Remarkably, SL12651and SL12653 yielded a transformation rate of up to 1×10⁻⁶ of DNA whengrown in M17G rich medium (FIG. 7A; +DNA). In contrast, the negativecontrol in absence of donor DNA had a spontaneous mutation rate of6×10⁻⁹ (FIG. 7A; −DNA). The transformants were validated by sequencingthe rpsL region covering the point mutation from the donor DNAconferring streptomycin resistance.

Then, the SL12653 strain was assayed in the same conditions withvariable quantity of donor DNA (0.5, 2.5, 5 and 25 μg mL⁻¹). It has beenshown that the transformation rate obtained is directly correlated tothe initial quantity of donor DNA, yielding up to a transformation rateof 5×10⁻⁶ (FIG. 7B).

Moreover, to confirm that the observed horizontal DNA transfer wasmediated by natural competence, the comX gene of SL12653 was knocked-out(as described in example 5 above). Three mutants of SL12653 withdisrupted comX gene were designed by inserting PCR products encompassingthe comX gene disrupted by the insertion of the chloramphenicolresistance cassette P₃₂-cat and validated by PCR for P₃₂-cat insertion.Transformation assays with rpsL* as donor DNA in all ΔcomX clones(ComX⁻) showed that the frequencies of appearance of Str^(r) clones weresimilar to the background level of spontaneous mutation frequencies(FIG. 7C). These results confirm that in SL12653, the transformation isdependent on the expression of the endogenous comX gene.

Finally, the transformability of the SL12653 strain was also assayed byoverexpressing the comX gene. Thus, an inducible comX expression plasmid[pGhPxylTcomX_(IO)] was constructed by cloning the comX gene from strainL. lactis subsp. lactis IO-1 under the control of the P_(xyIT) promoterfrom strain IO-1 on the thermosensitive plasmid pG⁺host9. This plasmidis a variant of pGhPxylTcomX_(MG) (pGIFPT001) described in David et al.,2017. The transformation procedure described in David et al (2017) wasfollowed. In presence of xylose (1%), SL12653 [pGhPxylTcomX_(IO)]yielded a transformation rate at least 20-fold higher than in absence ofxylose, confirming that the overexpression of comX in SL12653 increasedits transformability by natural competence.

Materials and Methods

Bacterial Strains, Plasmids, and Growth Conditions

The bacterial strains and plasmids used in this application are listedin Table 2.

TABLE 2 list of used bacterial strains and plasmids Strain or plasmidCharacteristics ^(a) Source or reference E. coli TG1 supE hsdΔ5 thiΔ(lac-proAB) Sambrook, J., E. F. Fritsch, and T. Maniatis. F′[traD36proAB⁺ lacl^(q) lacZΔM15] 1989. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. Km^(r), recA⁺; MC1000 containing a Law, J., G.Buist, A. Haandrikman, J. Kok, G. EC1000 copy of the repA gene frompWV01 Venema, and K. Leenhouts. 1995. J. in its chromosome Bacteriol.177: 7011-7018. L. lactis MG1363 Laboratory strain, dairy origin Gasson,M. J. 1983. J. Bacteriol. 154: 1-9. KW2 Wild-type isolate from cornKelly, W. J., E. Altermann, S. C. Lambie, and fermentation S. C. Leahy.2013. Front Microbiol. 4: 257. IL1403 Laboratory strain, dairy originChopin, A., M. C. Chopin, A. Moillo-Batt, and P. Langella. 1984. Plasmid11: 260-263 IO-1 Wild-type isolate from water in the Ishizaki A, OsajimaK, Nakamura K, Katsunori drain pit of a kitchen sink K, Hara T, andEzaki T. 1990. J. Gen. Appl. Microbiol., 36, 1-6 SL12651 Wild-typeisolate from plant DuPont/Danisco collection SL12653 material (maize)BLD101 KW2 kw2_0563::P_(comGA[MG])-luxAB This application BLD102 BLD101comEC::P₃₂-cat This application BLD107 BLD101 mecA::P₃₂-cat Thisapplication BLD108 BLD101 ciaRH::P₃₂-cat This application BLD109 BLD101covRS::P₃₂-cat This application BLD105 BLD101 clpC::P₃₂-cat Thisapplication L. raffinolactis LMG13098 Wild-type isolate from garden LMGcollection carrots LMG14164 Wild-type isolate from goose LMG collectionPlasmids pGEM ®-T easy Apr; cloning vector Promega pG⁺host9 Em^(r) TsMaguin, E., H. Prevost, S. D. Ehrlich, and A. Gruss. 1996. J. Bacteriol.178: 931-935 pGhost-Core Em^(r) Ts; pG⁺host9 derivative This applicationcontaining the Core part of the resolution site IRS recognized by theTnpl from Tn4430 pMG36eT Em^(r); E. coli-L. lactis shuttle vectorFontaine, L. and P. Hols. 2008. Appl. Environ. containing the P₃₂constitutive Microbiol. 74: 1102-1110. promoter from L. lactis pJIM4900Em^(r) Ts; pG⁺host9 derivative E. Guédon, (laboratory collection)containing the luxAB genes of Photorhabdus luminescens pXL Em^(r);pTRKH2 derivative containing Blomqvist T, Steinmoen H, Håvarstein L S.the luc reporter gene Appl Environ Microbiol. 2006. Oct; 72(10): 6751-6.pSEUDOPusp45GFP Em^(r); suicide vector containing the Overkamp, W., K.Beilharz, W. R. Detert llmg_pseudo_10(kw2_0563)::P_(usp45)- Oude, A.Solopova, H. Karsens, A. Kovacs, J. gfp⁺ insertion cassette Kok, O. P.Kuipers, and J. W. Veening. 2013. Appl. Environ. Microbiol. 79:6481-6490. pUC18Cm Ap^(r) Cm^(r): pUC18 derivative Goffin, P., F.Lorquet, M. Kleerebezem, and containing the P32-cat cassette P. Hols.2004. J. Bacteriol. 186: 6661-6666. pUC18Ery Ap^(r) Em^(r); pUC18derivative van Kranenburg, R., J. D. Marugg, I. I. van containing anerythromycin Swam, N. J. Willem, and W. M. de Vos. 1997. resistancemarker Mol. Microbiol. 24: 387-397. pNZ5319 Em^(r) Cm^(r): pACYC184derivative Lambert, J. M., R. S. Bongers, and M. containing the P32-catcassette Kleerebezem. 2007. Appl. Environ. Microbial. surrounded by loxsites 73: 1126-1135. pGhPcomGAluxAB Em^(r) Ts; pG⁺host9 derivative Thisapplication containing the llmg_pseudo_10(kw2_0563)::P_(comGA[MG])-luxAB insertion cassette pGhP32comX_(MG)Em^(r) Ts, pG⁺host9 derivative This application carrying comX of strainMG1363 under the control of the constitutive promoter P₃₂pGhP32comX_(IO) Em^(r) Ts, pG⁺host9 derivative This application carryingcomX of strain IO-1 under the control of the constitutive promoter P₃₂pGhP32comX_(MG)- pGhP32comX_(MG) derivative carrying This applicationP_(comGA[MG])-luc a P_(comGA[MG])-luc fusion pGhP32comX_(IO)-pGhP32comX_(IO) derivative carrying This application P_(comGA[IO])-luc aP_(comGA[IO])-luc fusion pGEMrpsL* Ap^(r), pGEM ®-T easy derivative Thisapplication carrying the rpsL* gene (strA1 allele) pUCcomECcat Ap^(r)Em^(r) Cm^(r), pUC18Ery derivative This application allowing theinsertion of P₃₂-cat at the comEC locus pGhPxylTcomX_(IO) Em^(r) Ts,pG⁺host9 derivative This application carrying comX of strain IO-1 underthe control of the inducible promoter P_(xylT) from IO-1 ^(a) Em^(r),Ap^(r), Cm^(r) and Ts: erythromycin, ampicillin, chloramphenicolresistance and thermo-sensitive RepA protein, respectively.

Escherichia coli was grown with shaking at 37° C. in Lysogeny-Broth (LB)broth. Plasmids derived from pMG36e and pG⁺host9 were constructed in E.coli strains TG1 and EC1000, respectively. L. lactis and L.raffinolactis were cultivated in M17 (Becton, Dickinson, and Company),Todd Hewitt broth (THB) (Becton, Dickinson, and Company) or CDM at 30°C. without agitation. M17 and THB were supplemented with 0.5% (w/v) ofglucose (M17G and THBG, respectively). Solid agar plates were preparedby adding 2% (w/v) agar to the medium. When required, 5 μg ml⁻¹ oferythromycin, 1 mg ml⁻¹ of streptomycin, and/or 10 μg ml⁻¹ ofchloramphenicol were added to the medium for L. lactis and L.raffinolactis; and 250 μg ml⁻¹ of erythromycin, 250 μg ml⁻¹ ofampicillin, 10 μg ml⁻¹ of chloramphenicol for E. coli.

Detection of Absorbance and Luminescence.

Growth (OD₆₀₀) and luciferase (Lux) activity were monitored at10-minutes intervals in a Varioskan Flash multi-mode reader(ThermoFisher). The luciferase activity is expressed in relative lightunits (RLU) and the specific luciferase activity in RLU OD₆₀₀ ⁻¹.

DNA Techniques and Electrotransformation

General molecular biology techniques were performed according to theinstructions given by Sambrook et al. (1989). Electrotransformation ofE. coli and L. lactis was performed as previously described. Theelectrotransformed cells of L. lactis were immediately resuspended in 1ml of M17G and incubated for 6 hours at 30° C. Chromosomal DNAs of L.lactis were prepared as previously described. PCRs were performed withPhusion DNA polymerase (NEB) in a GeneAmp PCR system 2400 (AppliedBiosystems). The primers used in this application are listed in Table 3.

TABLE 3 list of primers Primer name Sequence (5′-3′)Primers used for the construction of the constitutive comX expression plasmidpGhP32comX_(MG/IO): BID_ComXSDLLCup AAAAGAGCTCAATTATGAAAAAGAGGBID_ComXSDLLCdown AAAACTGCAGTTAATCATCATCTCG BID_ComXSDLLLupAAAAGAGCTCATAAAAGGAGAACTTTCC BID_ComXSDLLLdown AAAACTGCAGTCACTCTTCGTCTTCBID_pMGP32UpMfeI ATATCAATTGGTCCTCGGGATATGATAAG BID_pMGTerDownGACTTTGAACCTCAACTCCPrimers used for the construction of the P_(comGA[MG])-luxAB reporter strain BLD101:BID_LuxLLCf1 ATAGTCTCGAGTTTAAGCAATTGAATCGCTAG BID_LuxLLCr1GCAAAAAGTTTCCAAATTTCATACTAGAATATACGCAATTTG BID_LuxLLCf2CAAATTGCGTATATTCTAGTATGAAATTTGGAAACTTTTTGC BID_LuxLLCr2GCGAAAGGATCCCTATTAGGTATATTCCATGTGG BID_P3pseudoLLCGCTCCCTCGAGGGCGGCTCTGTTGGATTAATATATGGPrimers used for the construction of portable luc reporter vectors:BID_LucLLCr1 CTTTATGTTTTTGGCGGATCTCATACTAGAATATACGCAATTTG BID_LucLLCf2CAAATTGCGTATATTCTAGTATGAGATCCGCCAAAAACATAAAG BID_LucLLCr2GCGAAAGGATCCTTACAATTTGGGCTTTCCG BID_PcomGALLCF1*AAAACCCGGGTTTAAGCAATTGAATCGCTAG BID_PcomGALLLF1* 5′AAAACCCGGGAAATAAATGGCTACAAAATT BID_lucR1* AAAACGGCCGTTACAATTTGGGCTTTCCGBID_luxLLLf1 ATAGTCTCGAGAAATAAATGGCTACAAAATT BID_lucLLLr1CTTTATGTTTTTGGCGGATCTCATACTAGACTATACGCAAATAATC BID_lucLLLf2GATTATTTGCGTATAGTCTAGTATGAGATCCGCCAAAAACATAAAG BID_lucLLLr2GCGAAAGGATCCTTACAATTTGGGCTTTCCGPrimers used for the construction of pGhost-Core DD-pGhost-CoreUpAGCTTCCTAATACAACACAATTAATATTGTGTTGTATTATTG DD-pGhost-CoreDWAATTCAATAATACAACACAATATTAATTGTGTTGTATTAGGAPrimers used for rpsL sequencing: RpsL Univ UP ATGCCTACAATTAACCAATRpsL Univ DN CACCGTATTTAGAACGG LR_RpsL Univ UP ATGCCTACTATTAACCAATLR_RpsL Univ DN TACCGTATTTAGAACGG Primers used for rpsL amplification:BID_LLcdacARpsL AGTAGTATCAGCACTGACAGC BID_LLIcfusARpsLACACCTTTGTTCTTGAAGGprimers used for the construction of the comEC disruption mutant:BID_ComECLLCUp AAAGAGCTCAAAATAAAAATGAAATTATGG BID_ComECLLCDownAAAGCTAGCGGGAAAAAATTGTGAATTAC BID_CatUpSpeIAAAAACTAGTGCAGTTTAAATTCGGTCCTCGG BID_CatDownSpeIAAAAACTAGTGTACAGTCGGCATTATCTCATPrimers used for the construction and validation of the mecA deletion mutant:BID_fgt01FmecArec CTTTAATGATGGAATGATTG BID_fgt01RVmecArecCTATTAATCTTATCATATCCCGAGGATCCATATAACTATATGAAACC BID_fgt02FcatTCCTCGGGATATGATAAGATTAATAG BID_fgt02RVcat TCTCATATTATAAAAGCCAGTCATTAGBID_fgt03FmecArec CTAATGACTGGCTTTTATAATATGAGACTTAGAAAAATCTAAATATGGTTGBID_fgt03RVmecArec GAAGATTTTTAATTTCAAGTGTAG BID_mecAKOFTCAGTACCGAAAAACGAATG BID_mecAKORV ATTTACCAGTTCCGTTAGGPrimers used for the construction and validation of the ciaRH deletion mutant:BID_ciaRHUPF TAACAATGATACAGAAGATG BID_ciaRHUPRVRecCTATTAATCTTATCATATCCCGAGGATATTTTTGTCTTGTACTAGG BID_fgt02FcatTCCTCGGGATATGATAAGATTAATAG BID_fgt02RVcat TCTCATATTATAAAAGCCAGTCATTAGBID_ciaRHDownFRec CTAATGACTGGCTTTTATAATATGAGAGAGAGAAAAAAATTACTGACBID_ciaRHDownRV AAAATCTGTTAGAACTGTTG BID_ciaRHKODiagFAAGATAAGGCAGTTGAAATG BID_ciaRHKODiagRy TCACCATGTGAATAAAGTCCPrimers used for the construction and validation of the covRSdeletion mutant:BID_covRSfgt01F CAAAAATGTGAAGCTTATC BID_covRSfgt01RVRecCTATTAATCTTATCATATCCCGAGGATGCATAATTCGATTTC BID_fgt02FcatTCCTCGGGATATGATAAGATTAATAG BID_fgt02RVcat TCTCATATTATAAAAGCCAGTCATTAGBID_covRSfgt03FRec TAATGACTGGCTTTTATAATATGAGACTATTTATCTGCTCATTTCBID_covRSfgt03RV GAGCTTTTTTCAAATCTTC BID_covRSKOFdiag GAAGTGATGAATGAGATGBID_covRSKORVdiag CTTTCTCATCAATTGAGACPrimers used for the construction and validation of the clpC deletion mutant:BID_clpCUPF CTTTGGGTTCTAATTTATC BID_clpCUPRVRecCTATTAATCTTATCATATCCCGAGGACGTTGGTGTATATTTTAC BID_fgt02FcatTCCTCGGGATATGATAAGATTAATAG BID_fgt02RVcat TCTCATATTATAAAAGCCAGTCATTAGBID_clpCDownFRec CTAATGACTGGCTTTTATAATATGAGATAGAAATAAAGGAAAGGACBID_clpCDownRV TTGCTTTAAGGATAGTTTC BID_clpCFdiag AGAAGCCAATAATGACGATGBID_clpCRVdiag AGAATTCTGATGATGCACAGTCPrimers used for the construction of the inducible comX expression plasmid pGhPxylTcomX_(IO):FT_ AGCGCCGCGGTGGGATCCTCTAGAGTC pGhPxylcomXIOsacllrv FT_pGhPxylcomXCTGCAGGCATGCACATCATCAACTTGAAGGG FT_PxylTIOsacllfwCCCACCGCGGTGGAGATACGAACAAATTAG FT_PxylTIOrv GATAGTAACTCCTTAATTTTTATTTGCFT_comXIOrecfw GCAAATAAAAATTAAGGAGTTACTATCATGACATATTACTTGGAAGAAGAGGATTTTG FT_comXIOrecrv CCTTCAAGTTGATGATGTGCATGCCTGCAGTCACTCTTCGTCTTCPrimers used for the construction and validation of the SL12653-comX deletion mutantFT_comXlocusfw TGACCATGTTACACAAGCCTATATCCT FT_comXrecrvCGCCCTTATGGGATTTATCTTCCTTACTTCGTTTCTTTGCATAACTTCGTCTTA AT Uplox66TAAGGAAGATAAATCCCATAAGG Dnlox71 TTCACGTTACTAAAGGGAATGTA FT_comXrecfwTCTACATTCCCTTTAGTAACGTGAACCATGACCATTTTATAGGTTTAGATGTTT ATGAR_comxDNspecR CGGTGTTCCTCCATATATCTACGC FT_PxylcomXfwCGCTAAACTCAACAGGTGATCCGATTG

Construction of Plasmid pGhP32comX_(MG)

As a representative of the cremoris subspecies, the comX gene from thelaboratory strain MG1363 was initially chosen. ComX proteins of thissubspecies are highly conserved with at least 98% of identity. The comXgene was amplified by PCR using primersBID_ComXSDLLCup/BID_ComXSDLLCdown and inserted into plasmid pMG36eTunder the control of the constitutive P₃₂ promoter by SacI/PstI cloning,yielding plasmid pMGP32comX_(MG). The P₃₂-comX_(MG) fusion frompMGP32comX_(MG) was amplified by PCR with primersBID_pMGP32UpMfeI/BID_pMGTerDown, digested by MfeI/KpnI, and cloned inthe EcoRI/KpnI-digested thermosensitive pG⁺host9 vector. The resultingplasmid was named pGhP32comX_(MG).

Construction of Plasmid pGhP32comX_(IO)

As a representative of the lactis subspecies, the comX gene from theIO-1 strain was chosen. The comX gene was amplified by PCR using primersBID_ComXSDLLLup/BID_ComXSDLLLdown and inserted into plasmid pMG36eTunder the control of the constitutive P₃₂ promoter by SacI/PstI cloning,yielding plasmid pMGP32comX_(IO). The P₃₂-comX_(IO) fusion frompMGP32comX_(IO) was amplified by PCR with primersBID_pMGP32UpMfeI/BID_pMGTerDown, digested by MfeI/KpnI, and cloned inthe EcoRI/KpnI-digested thermosensitive pG⁺host9 vector. The resultingplasmid was named pGhP32comX_(IO).

Construction of Plasmid pGhost-Core

The Core part of the resolution site (IRS) recognized by the TnpIrecombinase from Tn4430 was assembled by using the complementary primersDD-pGhost-CoreUp/DD-pGhost-CoreDW. The resulting DNA fragment was clonedbetween HindIII and EcoRI sites in plasmid pG⁺host9. The resultingplasmid, named pGhost-Core, was transformed in E. coli harbouringplasmid pGIV004 (TnpI⁺) for obtaining multimeric forms (Vanhooff V,Galloy C, Agaisse H, Lereclus D, Revet B, Hallet B. Mol Microbiol. 2006May; 60(3):617-29).

Construction of P_(comGA[MG])-luxAB Reporter Strain BLD101

The P_(comGA[MG]) promoter was amplified by PCR from chromosomal DNA ofL. lactis MG1363 (identical nucleotide sequence between MG1363 and KW2)with primers BID_LuxLLCf1/BID_LuxLLCr1 (PCR1 product). The luxAB geneswere amplified by PCR from plasmid pJIM4900 with primersBID_LuxLLCf2/BID_LuxLLCr2 (PCR2 product). The P_(comGA[MG])-luxAB fusionwas created by overlapping PCR using PCR1 and PCR2 products and primersBID_LuxLLCf1/BID_LuxLLCr2. The resulting fusion was cloned in plasmidpSEUDOPusp45GFP using restriction enzymes XhoI and BamHI, yieldingplasmid pSEUDOPusp45PcomGAluxAB. In order to remove the P_(usp45)promoter, the entire vector except the P_(usp45) promoter was amplifiedby inverse PCR with primers BID_P3pseudoLLC/BID_LuxLLCf1 andself-ligated after XhoI digestion, leading to plasmid pSEUDOPcomGAluxAB.The insertion cassette llmg_pseudo_10::P_(comGA[MG])-luxAB was excisedfrom plasmid pSEUDOPcomGAluxAB and cloned into the pG⁺host9thermosensitive vector using restriction enzymes KpnI/EagI. Theresulting plasmid pGhPcomGAluxAB was then electro-transformed in strainKW2 and used to integrate the P_(comGA[MG])-luxAB cassette at locuskw2_0563 (llmg_pseudo_10 in MG1363) by double homologous recombination,resulting in the reporter strain KW2 kw2_0563::P_(comGA[MG])-luxAB(strain BLD101).

Construction of Portable Luc Reporter Systems

The P_(comGA[MG]) promoter was amplified by PCR from chromosomal DNA ofL. lactis MG1363 with primers BID_LuxLLCf1/BID_LucLLCr1 (PCR1 product).The luc gene was amplified by PCR from plasmid pXL with primersBID_LucLLCf2/BID_LucLLCr2 (PCR2 product). The P_(comGA[MG])-luc fusionwas created by overlapping PCR using PCR1 and PCR2 products and primersBID_LuxLLCf1/BID_LucLLCr2. The resulting fusion was cloned in plasmidpSEUDOPusp45GFP using restriction enzymes XhoI and BamHI, yieldingplasmid pSEUDOPusp45PcomGAluc. In order to remove the P_(usp45)promoter, the entire vector except the P_(usp45) promoter was amplifiedby inverse PCR with primers BID_P3pseudoLLC/BID_LuxLLCf1 andself-ligated after XhoI digestion, leading to plasmid pSEUDOPcomGAluc.The reporter cassette P_(comGA[MG])-luc was amplified by PCR frompSEUDOPcomGAluc (primers BID_PcomGALLCF1*/BID_IucR1*) and cloned betweenXmaI and EagI into the pGhP32comX_(MG) plasmid. The resulting reporterplasmid was named pGhP32comX_(MG)-P_(comGA[MG])-luc.

The P_(comGA[IO]) promoter was amplified from the IO-1 chromosome(primers BID_IuxLLLf1/BID_IucLLLr1) and the luciferase gene (luc) wasamplified from plasmid pXL (primers BID_IucLLLf2/BID_IucLLLr2). Thecassette P_(comGA[IO])-luc was created by overlapping PCR with primersBID_IuxLLLf1/BID_IucLLLr2. The cassette P_(comGA[IO])-luc was thenamplified from the overlapping PCR product with primersBID_PcomGALLLF1*/BID_IucR1* for XmaI/EagI cloning into pGhP32comX_(IO).The resulting reporter plasmid was namedpGhP32comX_(IO)-P_(comGA[IO])-luc.

Isolation of a rpsL Mutant Conferring Resistance to Streptomycin

Spontaneous streptomycin-resistant MG1363 clones were isolated on 1 mgml⁻¹ streptomycin-containing plates. After the sequencing of the rpsLgene with primers RpsL Univ UP/RpsL Univ DN, one spontaneous mutantresulting in a mutation (K56I) into the ribosomal protein S12 that waspreviously shown to confer resistance to streptomycin was selected (FIG.6). A 3.7-kb fragment containing the rpsL mutated gene (strA1 allele)was amplified by PCR with primers BID_LLcdacARpsL/BID_LLIcfusARpsL andcloned into the pGEM®-T easy vector (Promega), yielding plasmidpGEMrpsL*. This plasmid was used as template to generate the 3.7-kb PCRproduct with primers BID_LLcdacARpsL/BID_LLIcfusARpsL that was used asdonor DNA in natural transformation assays of strain KW2.

Standard Natural Transformation Assay

The BLD101 reporter strain carrying the pGhP32comX_(MG) plasmid (BLD101[pGhP32comX_(MG)]) was grown overnight in M17G at 30° C. Then, 1.5 ml ofthe pre-culture was diluted in 8.5 ml of fresh M17G medium to restartthe culture. After 2 hours of growth, cells were washed twice indistilled water and OD₆₀₀ was adjusted to 0.05 in CDM containingerythromycin (5 μg ml⁻¹) and supplemented with either 5% (v/v) glycerolor 5% (w/v) mannitol used as potential osmo-stabilizers. Typically, 5 μgof DNA was added in 300 μl of inoculated medium and the culture wasfurther incubated during 6 hours at 30° C. Cells were then spread onM17G agar plates supplemented with appropriate antibiotics and CFUs werecounted after 48 hours of incubation. The transformation frequency wascalculated as the number of antibiotic-resistant CFU ml⁻¹ divided by thetotal number of viable CFU ml⁻¹. In the case of streptomycin-resistanttransformants, antibiotic-resistant CFU ml⁻¹ corresponds to the numberof transformants obtained in presence of DNA less the number ofspontaneous transformants obtained in conditions where no DNA is addedin the culture. The transfer of the mutation conferring streptomycinresistance was confirmed by DNA sequencing of the rpsL gene after itsamplification by PCR using primers RpsL Univ UP/RpsL Univ DN.

Disruption of comEC by Natural Transformation

A comEC-containing DNA fragment of ˜3.2 kb was amplified by PCR withprimers BID_ComECLLCUp/BID_ComECLLCDown. Then, the PCR product wasdigested by SacI/NheI and cloned into the SacI/XbaI-digested suicideplasmid pUC18Ery (van Kranenburg et al., 1997), yielding plasmidpUCcomEC. To generate a comEC disruption cassette that allows theselection of double crossing-over recombinants, the P₃₂-cat fusionconferring resistance to chloramphenicol was cloned in the middle of thecomEC gene. For this purpose, the P₃₂-cat cassette was amplified by PCRfrom plasmid pNZ5319 (Lambert et al., 2007, Appl. Environ. Microbiol.73:1126-1135) with primers BID_CatUpSpeI/BID_CatDownSpeI. Theamplification product was digested by SpeI and cloned into theXbaI-digested pUCcomEC, yielding plasmid pUCcomECcat. This suicideplasmid was used to generate high quantity of donor DNA by PCRamplification for comEC disruption by natural transformation. Theinsertion of the P₃₂-cat cassette in the comEC gene of KW2 transformantswas validated by PCR (primers in Table 3).

Deletion of mecA, ciaRH, covRS, and clpC Genes by Natural Transformation

The mecA, ciaRH, covRS, and clpC genes were similarly inactivated by theexchange of their ORFs by the P₃₂-cat cassette using doublecrossing-over events. For this purpose, overlapping PCR productscontaining the P₃₂-cat cassette flanked by two recombination arms of˜1.5 kb (upstream and downstream homologous regions) were generated aspreviously reported. Briefly, upstream, downstream, and P₃₂-catfragments were separately amplified by PCR, purified, mixed in equimolarconcentration, and assembled by overlapping PCR by using the mostexternal primers (see list of primers in Table 3). 5 μg of the obtainedoverlapping PCR product was used as donor DNA for natural transformationof strain BLD101 [pGhP32comX_(MG)]. The correct insertion of the P₃₂-catcassette in each targeted locus of the KW2 transformants was validatedby PCR (see list of primers in Table 3). To obtain the final mutantstrains, the thermosensitive vector pGhP32comX_(MG) was cured by growingthe strains overnight at 37° C. without erythromycin. The cultures weresubsequently diluted and plated on M17G agar without erythromycin at 30°C. The resulting colonies were streaked in parallel on M17G plates withand without erythromycin. Absence of plasmid pGhP32comX_(MG) in Ery^(S)clones was validated by PCR.

Induction of Natural Competence in Lactococcus raffinolactis

Wild-type Lactococcus raffinolactis (i.e., L. raffinolactis strainswhich have not been previously engineered for the overproduction of thecomX gene) were grown overnight in M17G at 30° C. 1.5 ml of thepre-culture was diluted in 8.5 ml of fresh M17G medium to restart theculture. After 2 hours of growth, cells were washed twice in distilledwater and OD₆₀₀ was adjusted to 0.05 in CDM supplemented with either 5%(v/v) glycerol or 5% (w/v) mannitol used as potential osmo-stabilizers.15 μg of plasmid pGhost-Core was added in 300 μl of inoculated mediumand the culture was further incubated during 6 hours at 30° C. Cellswere then spread on M17G agar plates supplemented with appropriateantibiotics and CFUs were counted after 48 hours of incubation. Thetransformation frequency was calculated as the number ofantibiotic-resistant CFU ml⁻¹ divided by the total number of viable CFUml⁻¹.

Natural Competence in Lactococcus lactis Subsp Lactis SL12651 andSL12653 Strains

The L. lactis subsp. lactis SL12653 and 12651 strains were grownovernight at 30° C. Cells were washed twice in distilled water and OD₆₀₀was adjusted to 0.05 in M17G. Typically, 5 μg of donor DNA was added in200 μl of inoculated medium (25 μg/ml) and the culture was furtherincubated during 24 hours at 30° C. Cells were then spread on M17G agarplates supplemented with appropriate antibiotics and CFUs were countedafter 48 hours of incubation at 30° C. The transformation frequencycalculated exactly as described above (see Standard naturaltransformation assay).

The same experiments were done in SL12653 with various concentrations ofdonor DNA (0.5, 2.5, 5 and 25 μg/ml)

Construction of Plasmid pGhPxylTcomXIO

As a representative of the lactis subspecies, the comX gene and thepromoter of the xylT gene from the IO-1 strain were chosen. The comXgene was amplified by PCR using primers FT_comXIOrecfw andFT_comXIOrecry (PCR1), both containing overlapping sequences. The xylTpromoter region was amplified by PCR using primers FT_PxylTIOsacllfw andFT_PxylTIOrv (PCR2). The carrying vector was amplified from plasmidpGhP32comX_(MG) and amplified by PCR using primersFT_pGhPxylcomXIOsacllrv and FT_pGhPxylcomX (PCR3). The three PCRproducts were purified, mixed in an equimolar concentration andassembled by overlapping PCR using the most external primers, containinga SacII restriction site. The amplification product was digested by SacII and self-ligated. The resulting plasmid was named pGhPxylTcomX_(IO).

Transformation Assay in SL12653 Mutants Deleted for the comX Gene

The comX gene of SL12653 was inactivated by exchange of their ORF by theP₃₂-cat cassette using double crossing-over events. For this purpose,overlapping PCR products containing the P₃₂-cat cassette flanked by tworecombination arms of ˜1.5 kb (upstream and downstream homologousregions) were generated as previously reported. Briefly, upstream,downstream, and the P₃₂-cat fragments were separately amplified by PCR,purified and mixed in equimolar concentration, and assembled byoverlapping PCR by using the most external primers (see primers in Table3). 5 μg of the obtained PCR product was used as donor DNA for naturaltransformation of strain SL12653 [pGhPxylTcomX_(IO)] (ComX⁺). Thecorrect insertion of the P₃₂-cat cassette in the targeted locus ofSL12653 transformants was validated by PCR (see primers in Table 3). Toobtain the final mutant strains, the thermosensitive vectorpGhPxylTcomX_(IO) was cured by growing the strains overnight at 37° C.without erythromycin.

The cultures were subsequently diluted and plated on M17G agar withouterythromycin at 30° C. The resulting colonies were streaked in parallelon M17G plates with and without erythromycin. Absence of plasmidpGhPxylTcomX_(IO) in Ery^(S) clones was validated by PCR. Thus, 3 ΔcomXclones of SL12653 were obtained.

Xylose-Induced Natural Transformation in SL12653.

The L. lactis subsp. lactis SL12653 [pGhPxylTcomX_(IO)] was grownovernight at 30° C. Cells were washed twice in distilled water and OD600was adjusted to 0.05 in M17 supplemented with 1% (w/v) xylose.Typically, 5 μg of DNA was added in 200 μl of inoculated medium and theculture was further incubated during 24 hours at 30° C. Cells were thenspread on M17G agar plates supplemented with appropriate antibiotics andCFUs were counted after 48 hours of incubation at 30° C. Thetransformation frequency was calculated exactly as described above (seeStandard natural transformation assay).

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed present invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the present invention.Although the present invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, biochemistry, microbiology, bacteriology, or relatedfields are intended to be within the scope of the following claims.

REFERENCES

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1. A method for transforming a strain of the Lactococcus genus with anexogenous DNA polynucleotide comprising the steps of: (a) providing astrain of the Lactococcus genus, wherein said strain is transformablethrough natural competence; (b) modulating the production of a ComXprotein in said strain; (c) contacting said strain of step (b) with anexogenous DNA polynucleotide in a medium and incubating the resultingmixture for integration of the exogenous DNA polynucleotide into thegenome of said strain; and (d) selecting a strain which has integratedthe exogenous DNA polynucleotide into its genome.
 2. A method accordingto claim 1, wherein the step of modulating the production of a ComXprotein is performed by expressing a comX gene in said strain orincreasing the expression of a comX gene in said strain.
 3. A methodaccording to claim 2, wherein said comX gene is an exogenous comX gene.4. A method according to claim 3, wherein said exogenous comX gene istransferred into said strain by conjugation, transduction, ortransformation.
 5. A method according to claim 2, wherein said comX geneis the endogenous comX gene of said strain.
 6. A method according toclaim 5, wherein the method comprises carrying out step (b) and thencarrying out step (c) or comprises carrying out step (b) and step (c)simultaneously.
 7. A method according to claim 1, wherein said ComXprotein has: the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22; an amino acidsequence having at least 90% identity to the amino acid sequence of SEQID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ IDNO:22; or an amino acid sequence having at least 90% similarity to theamino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20 or SEQ ID NO:22.
 8. A method according to claim 1,wherein said comX gene has: the nucleotide sequence of SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21; or anucleotide sequence having at least 90% identity to the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, or SEQ ID NO:21.
 9. A method according to claim 1, wherein saidmedium of step (c) is a chemically defined medium.
 10. A methodaccording to claim 1, wherein prior to step (c) said strain is incubatedin a pre-culture medium.
 11. A method according to claim 1, wherein saidstrain is incubated with the exogenous DNA polynucleotide for around 4-8hours at around 30° C. and said medium of step (c) is supplemented withan osmo-stablizer.
 12. A method according to claim 1, wherein saidstrain of the Lactococcus genus of step (a) is a strain of theLactococcus raffinolactis species or a strain of the Lactococcus lactisspecies.
 13. A method according to claim 1, wherein said exogenous DNApolynucleotide used in step (c) is obtained from a strain of the samespecies as the strain provided in step (a).
 14. A strain of theLactococcus genus obtained by the method of claim
 1. 15. A method foridentifying a strain of the Lactococcus genus which is transformablethrough natural competence comprising the steps of: (a) providing astrain of the Lactococcus genus; (b) transforming said strain with aplasmid expressing a comX gene having at least 90% identity to theendogenous comX gene of said strain; (c) contacting said strain obtainedin step (b) with an exogenous DNA polynucleotide encoding a marker genein a medium and incubating the resulting mixture for integration of theexogenous DNA polynucleotide into the genome of said strain; and (d)determining the rate of recombination events; wherein a rate of at least1×10⁻⁶ transformants per μg of DNA is indicative of a strain which istransformable through natural competence.
 16. A method according claim1, wherein said strain of step (a) is identified using a method foridentifying a strain of the Lactococcus genus which is transformablethrough natural competence comprising: (a) providing a strain of theLactococcus genus; (b) transforming said strain with a plasmidexpressing a comX gene having at least 90% identity to the endogenouscomX gene of said strain; (c) contacting said strain obtained in step(b) with an exogenous DNA polynucleotide encoding a marker gene in amedium and incubating the resulting mixture for integration of theexogenous DNA polynucleotide into the genome of said strain; and (d)determining the rate of recombination events; wherein a rate of at least1×10⁻⁶ transformants per μg of DNA is indicative of a strain which istransformable through natural competence.
 17. A method according toclaim 1, wherein said strain of step (a) is identified using assay A,which is performed as follows: i) providing a strain of the Lactococcusgenus; ii) transforming the strain with a plasmid expressing a comX genehaving at least 90% identity to the endogenous comX gene of the strain;iii) pre-culturing the transformed strain overnight in a complex mediumsupplemented with glucose; iv) diluting about 1.5 mL of the pre-culturein 8.5 mL of fresh medium; v) after 2 hr of further growth at 30° C.,washing the cells twice with distilled water and adjusting the OD₆₀₀ to0.05 in a chemically defined medium comprising 5 μg mL⁻¹ erythromycinand an osmo-stabilizer; vi) adding 5 μg of exogenous DNA polynucleotidebearing an antibiotic resistance gene to 300 al of the culture medium;vii) incubating the resulting culture for 6 hr at 30° C.; viii) platingthe cells onto agar plates comprising the complex medium supplementedwith glucose and antibiotic corresponding to the antibiotic resistancegene of the exogenous DNA polynucleotide and incubating for 48 hr; ix)counting the colony forming units and determining the transformationrate, wherein: the transformation rate equals the number ofantibiotic-resistance colony forming units per mL divided by the totalnumber of viable colony forming units per mL, and a transformation rateof at least 1×10⁻⁶ transformants per μg of DNA is indicative of a strainthat is transformable through natural competence.
 18. A method accordingto claim 1, wherein said ComX protein has the amino acid sequence of SEQID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ IDNO:22.
 19. A method according to claim 1, wherein said comX gene has thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, or SEQ ID NO:21.
 20. A method according to claim11, wherein the osmo-stabilizer is glycerol or mannitol.